Multivalent binding proteins

Abstract

Provided herein are multivalent proteins comprising a first region capable of binding to one or more effector proteins and a second region capable of binding to one or more target proteins. Also provided are expression cassettes and vectors comprising nucleic acids encoding the multivalent proteins. Also provided are recombinant cells comprising the expression cassettes and vectors. Also provided are uses of such multivalent proteins, nucleic acids and recombinant cells. Also provided are the multivalent proteins, nucleic acids and recombinant cells for use as medicaments.

Description

[0001] Multivalent Binding Proteins

[0002] The present invention provides multivalent proteins comprising a first region capable of binding to one or more effector proteins and a second region capable of binding to one or more target proteins. Also provided are expression cassettes and vectors comprising nucleic acids encoding the multivalent proteins. Also provided are recombinant cells comprising the expression cassettes and vectors. The invention also provides uses of such multivalent proteins, nucleic acids and recombinant cells. Also provided are the multivalent proteins, nucleic acids and recombinant cells for use as medicaments, particularly for use in treating autoimmune diseases, autoinflammatory diseases, allergies, cancer and inflammation.

[0003] BACKGROUND

[0004] The immune system is a finely tuned network designed to detect and eliminate pathogens while maintaining tolerance to self-antigens. Central to this balance are inhibitory checkpoints, such as Programmed Cell Death Protein 1 (PD-1) and B and T Lymphocyte Attenuator (BTLA), which are crucial regulators of immune responses. Dysregulation of these checkpoint receptors and recognition of self-antigens can lead to chronic inflammation and the development of autoimmune diseases.

[0005] PD-1 is an inhibitory receptor expressed on the surface of activated T cells, B cells, and myeloid cells. Upon binding to its ligands, PD-L1 or PD-L2, PD-1 transmits a negative signal that attenuates T cell receptor (TCR) signalling. This mechanism prevents overactivation of the immune system, thereby reducing tissue damage during immune responses. However, when PD-1 signalling is insufficient or absent, T cells may become overly active, leading to chronic inflammation and autoimmunity.

[0006] Similarly, BTLA is an inhibitory receptor found on various immune cells, including T cells, B cells, and dendritic cells. BTLA interacts with its ligand, Herpesvirus Entry Mediator (HVEM), transmitting inhibitory signals that modulate immune cell activation and cytokine production. BTLA plays a significant role in maintaining peripheral tolerance and preventing excessive immune responses.

[0007] Autoimmune diseases arise when the immune system mistakenly targets self-antigens, leading to chronic inflammation and tissue damage. The failure of inhibitory checkpoints like PD-1 and BTLA contributes to the breakdown of self-tolerance. For instance, genetic polymorphisms or functional deficiencies in PD-1 have been associated with autoimmune conditions such as systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), and type 1 diabetes. Similarly, alterations in BTLA signalling pathways have been linked to increased susceptibility to autoimmune diseases.

[0008] In inflammation, autoimmune disorders and after organ transplants, therapeutic strategies aim to enhance the inhibitory signals of checkpoints like PD-1 and BTLA to restore immune balance. Checkpoint agonist antibodies are designed to mimic the natural ligands of these receptors or enhance their signalling pathways. By activating PD-1 or BTLA, these antibodies can dampen aberrant immune responses, reduce inflammation, and alleviate symptoms of autoimmune diseases.

[0009] Recent therapeutic strategies have focused on modulating these pathways using checkpoint agonist antibodies. The strategy involves inducing signalling by the checkpoint receptors that will, in turn, suppress the activity of lymphocytes. Agonist antibodies engage with checkpoint receptors and (through a still unknown mechanism) drive checkpoint receptor signalling. However, existing strategies invariably rely on crosslinking of the agonist antibodies to induce signalling. Cross-linking is achieved in vitro by coupling the agonist antibodies to beads, glass or plastic surfaces. In vivo, cross-linking can be achieved by promoting interactions between the Fc component of the antibody with members of the Fc Receptor (FcR) family. Importantly, in the absence of cross-linking, agonist antibodies fail to induce signalling and suppress immune responses. A second mechanism of action of known PD-1 agonistic antibodies is the mediation of antibody-dependent cellular cytotoxicity or phagocytosis via NK or myeloid cells, respectively, resulting in the depletion of PD-1 expressing T cells, preferentially of T cells with high PD-1 expression levels. Whilst this strategy has been demonstrated to work in vitro and in preclinical models, a recent double-blind, randomized, placebo-controlled Phase 2 trial of an agonist antibody against PD-1 failed to show superiority over existing therapeutic strategies in RA. A recent Phase 2b study of a BTLA agonist antibody in atopic dermatitis also failed to meet its primary endpoint. While the results from these trials supported the targeting of inhibitory receptors for suppression of T cells in RA, they also highlighted the need for improved mechanistic modalities to trigger sustained and potent PD-1 signals, particularly without the need for external triggers such as the presence of FcyR expressing B or innate immune cells.

[0010] Previous work that leading to the development of agonist antibodies, particularly focusing on PD-1 and BTLA, enabled the identification of some molecular properties that appear to favour agonism. These properties include: membrane-proximal binding1, low binding affinity to target receptors with a fast off-rate2, and binding to Fc Receptors (FcR)1 3. Under this workflow, agonist antibodies will have to be developed to recognise specific receptors and engineered to recognize specific, membrane-proximal, epitopes. Furthermore, they become inert in the absence of Fc receptor binding. In fact, FcR-dependence poses additional challenges as FcR- driven activation of NK-cells or macrophages can result in the secretion of inflammatory cytokines and antibody dependent cell mediated cytotoxicity (ADCC) of receptor-positive host cells (“depletion”). These pro-inflammatory effects can be particularly damaging if it occurs in inflamed tissues. Moreover, in chronic disease settings, such as RA and SLE, prolonged exposure to agents that deplete T and / or B cells raises the concern that the immune response may become compromised and that depleting T and / or B cell clonotypes in the immune repertoire may weaken future responses against infection, for example.

[0011] Accordingly, there is a need to provide novel therapeutic tools for triggering said inhibitory receptors that do not rely on FcR binding. In addition, there is a need to provide novel agonism methods specifically targeting PD-1 , BTLA and other inhibitory receptors that does not rely on FcR binding. The present invention aims to address these needs at least in part.

[0012] BRIEF SUMMARY OF THE DISCLOSURE

[0013] This invention is based on the inventors’ surprising finding that multivalent (e.g. bispecific) molecules (e.g. polypeptides) that bind the cell surface receptors (such as the CD8 effector protein and the PD-1 inhibitory receptor) expressed on T cells drive phosphorylation of PD-1 , e.g., by bringing the CD8 effector protein into proximity with the PD-1 inhibitory receptor. They observed that this phosphorylation of PD-1 resulted in a signalling cascade that suppresses T cell activation and decreases cytokine expression from cells in the presence or absence of PD-L1 . The inventors have shown that the binding of both CD8 and PD-1 by multivalent proteins exerts a suppressive effect. Surprisingly, multivalent proteins recognising distinct epitopes on CD8 displayed T cell suppressive effects, which suggests that the activity can be tuned by using specific amino acid compositions. Furthermore, the inventors surprisingly observed that the multivalent proteins induced a stronger suppression effect than agonist antibodies of the state of the art. Moreover, the inventors’ multivalent proteins do not require an Fc domain or FcR binding, unlike the agonist antibodies of the state of the art.

[0014] In a first aspect there is provided a multivalent protein comprising: a. a first region capable of binding to one or more effector proteins, the effector protein comprising or associated with protein kinase activity; and b. a second region capable of binding to one or more target proteins, wherein the target protein signals through a phosphorylation mechanism. It will be understood that the multivalent proteins described herein may be bispecific (i.e. bind to one effector and one target protein), tri-specific (bind to one target and two effector proteins or two target and one effector protein), tetra-specific (bind to two target and two effector proteins) and so on.

[0015] In certain embodiments the first and second regions are operably linked.

[0016] In certain embodiments the multivalent protein comprises a linker interposed between the first and second regions.

[0017] In certain embodiments at least one of the first and / or second region each comprise a binding domain for binding to the effector and / or target proteins. In certain embodiments at least the first region comprises a binding domain for binding to the effector and / or target proteins. In certain embodiments at least the second region comprise a binding domain for binding to the effector and / or target proteins. In certain embodiments at least the first region comprise a binding domain for binding to the effector proteins. In certain embodiments at least the first region comprise a binding domain for binding to the target proteins. In certain embodiments at least the second region comprise a binding domain for binding to the effector proteins. In certain embodiments at least the second region comprise a binding domain for binding to the target proteins.

[0018] In certain embodiments, each binding domain comprises an antigen-binding domain or a protein-binding ligand, optionally wherein: a. the antigen-binding domain comprises an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VHdomain, a VLdomain, a single domain antibody (dAb), a VNAR domain, a VHH domain, a T cell receptor (TCR) binding subunit, an antibody mimetic such as a DARPin, anticalin, affibody, minibinder, cyclic or bicyclic peptide or a functional fragment of any thereof; and / or b. the protein-binding ligand comprises a cytokine, a growth factor, a receptor extracellular domain (ECD), a peptidic hormone, GPCR ligand or a functional variant of any thereof.

[0019] In certain embodiments, at least the first region comprise an antigen-binding domain or a protein-binding ligand as described herein. In certain embodiments, at least the second region comprise an antigen-binding domain or a protein-binding ligand as described herein.

[0020] In certain embodiments, the multivalent protein comprises at least one first polypeptide chain comprising at least a portion of the first and / or second region and at least one second polypeptide chain comprising at least a second portion of the first and / or second region. In certain embodiments, the multivalent protein comprises at least one first polypeptide chain comprising at least a portion of the first region. In certain embodiments, the multivalent protein comprises at least one first polypeptide chain comprising at least a portion of the second region. In certain embodiments, the multivalent protein comprises at least one second polypeptide chain comprising at least a second portion of the first region. In certain embodiments, the multivalent protein comprises at least one second polypeptide chain comprising at least a second portion of the second region.

[0021] In certain embodiments, the first region and / or the second region or a portion thereof each comprise at least one Fc region.

[0022] In certain embodiments, at least a portion of the first region and at least a portion of the second region are linked via the Fc regions or a portion thereof.

[0023] In certain embodiments, the first region and / or the second region or a portion thereof each comprise at least a portion of a TCR constant domain.

[0024] In certain embodiments, the multivalent protein comprises a multivalent antibody structure. In one embodiment, the multivalent antibody structure is a bispecific antibody, or antigen-binding portion thereof.

[0025] In certain embodiments, the multivalent antibody structure is selected from a single domain bispecific antibody, diabody, Dual-Affinity Re-Targeting molecule (DART), Tandem ScFv (BiTE), CrossMab, CrossMab-Fab, tandem CrossFab, TCR-like antibody, and tandem diabody or variants thereof.

[0026] In certain embodiments, the multivalent protein comprises a multivalent non-antibody based scaffold protein (i.e. an antibody mimetic) or a functional fragment thereof. DARPin, anticalin, affibody, minibinder cyclic or bicyclic peptide.

[0027] In certain embodiments, the multivalent protein comprises a TCR. In certain embodiments, the multivalent protein comprises TCR-like antibody.

[0028] In certain embodiments, the effector protein and / or target protein each comprise one or more extracellular domains. In certain embodiments, the effector protein comprises one or more extracellular domains. In certain embodiments, the target protein comprises one or more extracellular domains.

[0029] In certain embodiments, the effector protein and / or target protein each comprise one or more cell surface receptor proteins. In certain embodiments, the effector protein comprises one or more cell surface receptor proteins. In certain embodiments, the target protein comprises one or more cell surface receptor proteins.

[0030] In certain embodiments, the effector protein and target protein are present on the same cell. In certain embodiments, the multivalent protein binds the effector and target protein in cis.

[0031] In certain embodiments, the effector protein comprises a receptor-protein tyrosine kinase.

[0032] In certain embodiments, the effector protein comprises a protein that comprises one or more extracellular domains wherein the effector protein interacts with one or more intracellular protein kinases.

[0033] In certain embodiments, the effector protein is selected from CD4, CD8, CD48, CD2, CD59, CD55, CD24, and CD14.

[0034] In certain embodiments, the one or more intracellular protein kinases comprises one or more Src family kinases; optionally wherein the one or more Src family kinases comprise Src, Yes, Fyn, Lyn, Hck, Lek, Fgr, Blk, and / or Yrk.

[0035] In certain embodiments, the target protein mediates signalling through a tyrosine motif.

[0036] In certain embodiments, the tyrosine motif is selected from one or more of an ITAM, ITSM, or ITIM motif. In certain embodiments, the tyrosine motif is an intracellular motif that is chemically modified by a phosphorylation of a tyrosine residue.

[0037] In certain embodiments, the target protein comprises an immune-checkpoint receptor.

[0038] In certain embodiments, the target protein comprises a cytokine receptor.

[0039] In certain embodiments, the target protein comprises an interferon receptor.

[0040] In certain embodiments, the target protein comprises tumour necrosis factor receptor.

[0041] In certain embodiments, the target protein comprises a growth factor receptor.

[0042] In certain embodiments, the target protein comprises the target protein comprises an inhibitory receptor protein.

[0043] In certain embodiments, the inhibitory receptor protein is selected from

[0044] PD-1 , BTLA, CD5, CD6, CD200R, VISTA, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, CD66, LAIR1 , ILT-2, ILT-4, CD31 , SIRPa,, members of the CEACAM family, members of the SLAM family of surface receptors, and members of the KIR family of surface receptors. In certain embodiments, the inhibitory receptor protein is selected from PD-1 , BTLA, CD5, CD6, CD200R, VISTA, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, CD66, LAIR1 , ILT-2, ILT-4, CD31 , SIRPa. In certain embodiments, the inhibitory receptor protein is selected from members of the CEACAM family, members of the SLAM family of surface receptors, and members of the KIR family of surface receptors.

[0045] In certain embodiments, the multivalent protein comprises from N-terminal to C-terminal

[0046] Domain A comprising a binding region for binding to the effector protein; a linker; and

[0047] Domain B comprising a binding region for binding to the target protein.

[0048] In certain embodiments, the multivalent protein comprises from N-terminal to C-terminal

[0049] Domain A comprising a binding region for binding to the target protein; a linker; and

[0050] Domain B comprising a binding region for binding to the effector protein.

[0051] In certain embodiments, the multivalent protein comprises from N-terminal to C-terminal

[0052] Domain A comprising a first portion of a binding region for binding to the effector protein and a first portion of a binding region for binding to the target protein; a linker;

[0053] Domain B comprising a second portion of the binding region for binding to the effector protein and a second portion of the binding region for binding to the target protein.

[0054] In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD-1 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least PD- 1 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least BTLA and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD5 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD200R and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least VISTA and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM- 3 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM-3 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM-3 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM-3 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM- 3 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM-3 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM-3 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIM-3 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least TIGIT and CD55. TIGIT and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD22 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-7 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-14 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-9 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least Siglec-6 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD300a and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRIIB and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRIIB and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRHB and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRIIB and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRIIB and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRHB and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRIIB and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least FcyRIIB and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD45 and an activatory FcyR (e.g. FcyRI, FcyRIIA, or FcyRIH). In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least NKG2A and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD72 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA- 4 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CTLA-4 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD66 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least LAIR1 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-2 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT- 2 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-2 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-2 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-2 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT- 2 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-2 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-2 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-4 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT- 4 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-4 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-4 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-4 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT- 4 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-4 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least ILT-4 and CD59. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD2. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD4. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD8. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD14. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD24. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD48. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD55. In certain embodiments there is provided a multivalent protein that is capable of binding to at least CD31 and CD59.ln certain embodiments, the multivalent protein comprises a first region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 14, 15, 20, 21 , 22, 23, 24, 49, 50, 51 , 52 and a second region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1 , 5, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48.

[0055] In certain embodiments, the first region comprises an amino acid sequence having at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 2, 14, 15, 20, or 21 . In certain embodiments, the second region comprises an amino acid sequence having at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 1 , 5, 36, 37, or 45.

[0056] In certain embodiments, the multivalent protein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 122, 123, 124, or 125.

[0057] In certain embodiments, the multivalent protein comprises an amino acid sequence having at least 80%, 90%, 95% or 99% sequence identity to any one of SEQ ID NOs: 55, 57, 58, 59, 60, 61 , 65, 66, 79, 81 , 82, 83, 84, 86, 87, 88, 92, 93, 122, 124, or 127.

[0058] In a second aspect there is provided at least one nucleic acid molecule encoding a multivalent protein as described herein. In certain embodiments, the nucleic acid molecule is comprised within an expression cassette or vector.

[0059] In certain embodiments, there is provided a nucleic acid composition comprising one or more nucleic acid molecules each encoding at least a portion of a multivalent protein as described herein. In certain embodiments, the nucleic acid molecules are comprised within one or more expression cassettes or vectors.

[0060] In a third aspect there is provided a recombinant cell comprising the nucleic acid as described herein.

[0061] In a fourth aspect there is provided a method of producing a multivalent protein as described herein, the method comprising: providing a recombinant cell as described herein; and expressing the one or more nucleic acid molecules to produce the multivalent protein.

[0062] In a fifth aspect there is provided a pharmaceutical composition comprising a multivalent protein, at least one nucleic acid molecule, or a recombinant cell as described herein.

[0063] In a sixth aspect there is provided a method of treating a disease or condition in a subject, the method comprising administering an effective amount of a multivalent protein, at least one nucleic acid molecule, a recombinant cell, or a pharmaceutical composition as described herein to a subject in need thereof.

[0064] In a sixth aspect there is provided a method for modulating cell signalling mediated by and a target protein, wherein the target protein signals through a phosphorylation mechanism in a subject, the method comprising administering an effective amount of a multivalent protein, at least one nucleic acid molecule, a recombinant cell, or a pharmaceutical composition as described herein, to a subject in need thereof.

[0065] In a seventh aspect there is provided a multivalent protein, at least one nucleic acid molecule, a recombinant cell, or a pharmaceutical composition as described herein, for use as a medicament.

[0066] In certain embodiments, the multivalent protein, at least one nucleic acid molecule, the recombinant cell, or the pharmaceutical composition as described herein, are for use an adjuvant in prophylaxis or treatment of infection, autoimmunity, and / or cancer. The multivalent proteins described herein may help to guide immune subset responses and therefore act as an adjuvant in treatment of diseases such as infection, autoimmunity, and / or cancer.

[0067] In an eighth aspect there is provided a multivalent protein, at least one nucleic acid molecule, a recombinant cell, or a pharmaceutical composition as described herein, for use in treating: an autoimmune disease; inflammation; transplantation; allergies; or cancer.

[0068] In certain embodiments, the multivalent protein, at least one nucleic acid molecule, recombinant cell or pharmaceutical composition as described herein modulates an immune response in the subject.

[0069] In certain embodiments, the multivalent protein, at least one nucleic acid molecule, recombinant cell or pharmaceutical composition as described herein modulates inflammation in the subject.

[0070] In certain embodiments, the multivalent protein, at least one nucleic acid molecule, recombinant cell or pharmaceutical composition as described herein suppress T cell activation in the subject.

[0071] In certain embodiments, the multivalent protein, at least one nucleic acid molecule, recombinant cell or pharmaceutical composition as described herein enhance inhibitory signalling of checkpoint proteins in the subject. Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of them mean “including but not limited to”, and they are not intended to (and do not) exclude other moieties, additives, components, integers or steps.

[0072] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0073] Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.

[0074] Various aspects of the invention are described in further detail below.

[0075] BRIEF DESCRIPTION OF THE FIGURES

[0076] Embodiments of the invention are further described hereinafter with reference to the accompanying drawings, in which:

[0077] Figure 1 shows development of activatory Proximity ImmunoModulators (aProxIM) to drive activation of surface receptors, including inhibitory receptors such as PD-1 and BTLA, by the recruitment, in cis (on or in the same cell), of receptors directly connected to an intracellular kinase or associated, through a covalent bond or a transient interaction, with intracellular kinases.

[0078] Figure 2 shows: A and B, aProxIM treatment on Jurkat cells and WB: TCR-, CD8cx(3+ Jurkat T cells were transduced with 250 pl of lentivirus encoding wild-type PD-1 with an N- term HA tag. For pervanadate treatment, 6 million cells were transferred to a 6-well plate and treated with 1 mM (final cone) freshly prepared pervanadate, for 30 min at 37°C. 10 million cells per condition were harvested and washed 1x with PBS. Cells were treated with 500nM aProxIMI 0 or with PBS for 5, 15, 30, 45 and 60 min at 37°C. Subsequently, cells were lysed and total HA-tagged PD-1 (and interacting proteins) were recovered with an anti-HA IP. IP eluate was loaded on SDS-PAGE for WB. 3% BSA in PBS-T was used as a blocking solution. Antibody dilutions were prepared in Intercept Blocking Buffer. C aProxIM treatment on LentiX™ 293T cells and WB. LentiX™ 293T cells of 3 wells of 6-well plates were pooled 48h after transfection. A total of 1 .5 ug pDNA were transfected per well 48h post transfection, cells were harvested and incubated with 500 nM aProxIM or with PBS for 10 min on ice, followed by 20 min at 37°C. Subsequently, cells were lysed and an IP was done to recover HA-tagged protein (PD-1). 3% BSA in PBS-T was used as a blocking solution. Antibody dilutions were prepared in Intercept Blocking Buffer.

[0079] Figure 3A shows aProxIM treatment, surface IP and WB. LentiX™ 293T cells of 3 wells of 6-well plates were pooled 48h after transfection. A total of 1 .5 ug pDNA were transfected per well 48h post transfection, cells were harvested and incubated with 500 nM aProxIM 10 / 11 / 12, 500 nM aProxIM 10 / 11 / 12 treated with 3C protease over night or with PBS for 10 min on ice, followed by 20 min at 37°C. Cells were incubated with biotinylated anti-HA antibody for 1 h on ice. Subsequently, cells were washed once and lysed. An IP using magnetic SA beads was done to recover surface HA-tagged protein (PD-1). 3% BSA in PBS-T was used as blocking solution. Antibody dilutions were prepared in Intercept Blocking Buffer. Figure 3 B shows Coomassie-stained SDS-PAGE showing the 3C-cleavage of aProxIM 10 / 11 / 12. aProxIM samples were treated with 1 :100 (w / w ratio) 3C protease and cleaved overnight at 4°C.

[0080] Figure 4 A shows Jurkat Activation and aProxIM or Nivolumab treatment: A1 -K562 cells (+ / -PD-L1) were pulsed with 50 pM of MAGE-3 peptide for 2 hours. 100k A3A- Jurkat (+ / - PD- 1) were co-cultured with 50k pulsed K562 cells in 200 pL RPMI media with PBS or 1 nM aProxlM-12 or 10 pg / ml of Nivolumab. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, PD-1 -PE, PD-L1 - FITC, CD45-AF488, TCR-a / b-PE for 45 minutes and surface expression determined by flow cytometry. Median fluorescence intensity (MFI) for CD69 expression is shown. Figure 4 B shows Peptide titration and Jurkat Activation: A1-K562 cells (+ / - PD-L1) were pulsed with 0, 10, 50, 100 pM of MAGE-3 peptide for 2 hours. 100k A3A- Jurkat (+ / -PD-1) were co-cultured with 50k pulsed K562 cells in 200 pL RPMI media with FBS. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69- APC, PD-1 -PE, PD-L1-FITC, CD45-AF488, TCR-a / b-PE for 45 minutes and surface expression determined by flow cytometry.

[0081] Figure 5 shows Jurkat Activation and aProxIM treatment: A1 -K562 cells (+ / -PD-L1) were pulsed with 50 pM of MAGE-3 peptide for 2 hours. 100k A3A-Jurkat (+ / - PD-1) were co- cultured with 50k pulsed K562 cells in 200 pL RPMI media with PBS or 1 nM aProxlM-12. After 18 hours, 150 pL of supernatant were collected and 100 pL of the samples were added to each well for quantification of IL-8 in the supernatant by ELISA. Data below represent blank corrected raw absorbance values.

[0082] Figure 6 shows Jurkat Activation and aProxIM treatment: A1-K562 cells (-PD-L1) were pulsed with 0, 10, 50, pM of MAGE-3 peptide for 2 hours. 100k A3 A- Jurkat (+ PD-1) were co- cultured with 50k pulsed K562 cells in 200 pL RPMI media with FBS. They are treated with 10, 3, 1 , 0.3 nM of aProxlM-10 and 12. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, CD45-AF488. Data shows CD69 MFI.

[0083] Figure 7 A shows Jurkat Activation and aProxIM treatment: A1-K562 cells (-PD-L1) were pulsed with 50 pM of MAGE-3 peptide for 2 hours. 100k A3A-Jurkat (+ PD-1) were co-cultured with 50k peptide pulsed K562 cells in 200 pL RPMI media with FBS. They are treated with 30, 10, 3, 1 , 0.3, 0.1 , 0.03 nM aProxlM-8 or 10. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, PD-1 -PE, CD45- AF488. Data shows CD69 MFI. B Expression of CD69 (MFI and normalised fold changes) on A3A-Jurkat T cells (+ PD-1) after 18 hours of co-culture with A1-K562 (- PD-L1) at 2:1 ratio, with aProxlM-10, 11 , 12 treatment.

[0084] Figure 8 shows Jurkat Activation and aProxIM treatment: A1-K562 cells (-PD-L1) were pulsed with 50 pM of MAGE-3 peptide for 2 hours. 100k A3A-Jurkat (+ PD-1) were co-cultured with 50k peptide pulsed K562 cells in 200 pL RPMI media with FBS. They are treated with 30, 10, 3, 1 , 0.3, 0.1 , 0.03 nM aProxlM-12, 13, 17 or 18. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, PD- 1-PE, CD45-AF488. Data shows CD69 MFI.

[0085] Figure 9 shows Normalized fold changes of CD69 expression on PD-1 Jurkat-A3A cells (+ / - CD8) after 18 hours of co-culture with A1-K562 (- PD-L1) at 2:1 or 1 :1 ratio, with aProxlM-12 treatment. Jurkat Activation aProxIM treatment: A1-K562 cells (-PD-L1) were pulsed with 0, 50, 100 pM of MAGE-3 peptide for 2 hours. 100k PD-1 A3A-Jurkat (+ / - CD8) were co-cultured with 50k pulsed K562 cells in 200 pL RPMI media with FBS. They are treated with 30, 10, 3, 1 , 0.3, 0.1 , 0.03 nM of aProxlM-12. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, PD-1 -PE, CD45- AF488.

[0086] Figure 10 shows Jurkat Activation and aProxIM treatment: A1 -K562 cells (-PD-L1) were pulsed with 50 pM of MAGE-3 peptide for 2 hours. 100k A3A-Jurkat (+ PD-1) were co-cultured with 50k pulsed K562 cells in 200 pL RPMI media with FBS. They were treated with 100, 30, 10, 3, 1 , 0.3, 0.1 , 0.03 nM of aProxlM-12 (+ / - 3C protease, “3C-digested”, left western blot). After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, PD-1 -PE, CD45-AF488. Data shows normalized fold changes of CD69 expression (right).

[0087] Figure 11 shows aProxIM treatment on Jurkat T cells and WB. TCR-, CD8cx(3+ Jurkat cells were transduced with 250 pl of PD-1 -HA, PD-1 or (YF)-HA, lentivirus. 20 million cells per condition were harvested and washed Ixwith PBS. Cells were treated with 500nM aProxIM or with PBS for 5, 15 or 25 min at 37°C. Subsequently, cells were lysed and total HA-tagged PD-1 (and interacting proteins) were recovered with an anti-HA IP. IP eluate was loaded on SDS-PAGE for WB. 3% BSA in PBS-T was used as a blocking solution. Antibody dilutions were prepared in Intercept Blocking Buffer.

[0088] Figure 12 shows Expression of CD69 (MFI, top) and fraction of CD69+ T cells (bottom) on A3A-Jurkat T cells (+ PD-1) after 18 hours of co-culture with A1-K562 (- PD-L1) at 2:1 ratio, with or without aProxIM treatment. Jurkat Activation and aProxIM treatment: A1-K562 cells (- PD-L1) were pulsed with 0 or 50 pM of MAGE-3 peptide for 2 hours. 100k A3A- Jurkat (+ PD- 1) were co-cultured with 50k pulsed K562 cells in 200 pL RPMI media with FBS. They were treated with 100, 30, 10, 3, 1 , 0.3, 0.1 , 0.03 nM of aProxlM-12 or Clone-19. After 18 hours, the cells were washed and stained with Zombie Violet for 15 minutes, followed by antibodies against CD69-APC, PD-1 -PE, CD45-AF488.

[0089] Figure 13 shows Jurkat T cell activation and BTLA aProxIM treatment. A 27 million Jurkat cells transduced with 1 G4 TCR, CD8cx(3 and BTLA were spun down per condition. Jurkat cells were washed with PBS and treated with 50 nM or 500 nM BTLA-aProxlM-02 (BTLA scFv 2.8.6 - CD8 VHH21), with PBS or with 500 nM BTLA-aProxlM-02 cleaved with 3C protease (“+3C”). After treatment, cells were lysed. HA-tagged protein (BTLA) and interacting proteins were recovered by immunoprecipitation using magnetic anti-HA beads. Mild conditions were used for lysis and IP (using DDM detergent for membrane solubilization and one washing step). Antibodies used in western blot: Rat anti-HA (1 :4000), Goat anti-rat 800CW (1 :5000); Mouse anti-pY100 (1 :4000), Donkey anti-mouse 680RD (1 :5000). Antibodies blot2: Rat anti-HA (1 :4000), Goat anti-rat 680RD (1 :5000); Rabbit anti-SHP2 (1 :4000); Donkey anti-rabbit 800CW (1 :5000). B Coomassie-stained SDS-PAGE showing the cleavage of BTLA- aProxlM-02 with 3C protease. C Jurkat T cell activation and aProxIM treatment: 100.000 1 G4 TCR+, CD8cx(3+ Jurkat T cells (+ / - BTLA) were co-cultured with 50.000 THP1s (9V HLA- A02:01 , single-chain trimer) and treated with 0, 0.15, 0.46, 1 .3, 4, 12, 37, 111 , 333, 1000 nM BTLA-aProxlM-02. After 18h , cells were stained with viability stain Zombie Violet, CD69-APC, and HLA-A02-FITC.

[0090] Figure 14 shows aProxlM-12 and aProxlM-13 activity in a mixed lymphocyte reaction (MLR). Human CD3+T cells (100,000 per well), isolated from healthy donor PBMCs, were co- cultured with 10,000 allogeneic monocyte-derived dendritic cells in 200 pL total volume for 5 days. T-cell activation was assessed by flow cytometry following staining for CD3, CD4, CD8, CD25, Ki-67, and PD-1 . Cells were treated with 200 nM aProxlM-12 (CD8xPD-1) or aProxIM- 13 (CD4xPD-1). As benchmark controls, PD-1 agonist antibodies Rosnilimab or Clone-19 were used at 10 pg / mL. (A, B) Data gated on CD3+CD4+T cells showing the percentage of CD25+(A) or Ki-67+(B) cells. (C, D) Data gated on CD3+CD8+T cells showing the percentage of CD25+(C) or Ki-67+(D) cells. aProxlM-12 and aProxlM-13 each selectively suppress activation markers in the T-cell subset expressing their corresponding effector-arm target (CD8 foraProxlM-12; CD4 foraProxlM-13), demonstrating subset-restricted inhibitory activity.

[0091] Figure 15 shows CD200R-aProxlM treatment on HEK293 LentiX cells. HEK293 LentiX cells were transfected with a total of 1 .1 pg plasmid DNA per well encoding CD200R, CD8a / p, and Lek Y505F. At 48 hours post-transfection, cells from three wells were pooled and washed with PBS. (A) Flow cytometry analysis. Cells were stained with anti-CD200R-APC (1 :200; BioLegend) and anti-CD8a-AF488 (1 :200; BioLegend) to assess surface expression of CD200R and CD8. (B) RAKL stimulation and immunoprecipitation. Cells were treated with 5 nM, 50 nM, or 500 nM hCD200R-aProxim-01 , or with PBS control, for 10 min on ice followed by incubation at 37 °C for 5 or 15 min. Cells were lysed and HA-tagged CD200R was recovered by anti-HA immunoprecipitation. Eluted material was resolved by SDS-PAGE and analysed by western blot. Intercept Blocking Buffer was used for membrane blocking, and antibody dilutions were prepared in Intercept Blocking Buffer (anti-HA 1 :4000; Roche; anti- pY1000 1 :1000; Cell Signaling Technology). Membranes were imaged on a LI-COR Odyssey system. Treatment with hCD200R-aProxim-01 resulted in reduced phosphorylation of CD200R.

[0092] Figure 16 shows that BTLA aProxIM molecules dampen T-cell activation. (A) Jurkat T cell activation for cells expressing BTLA and treated with a BTLA aProxIM dose-response. Jurkat T cells expressing the 1 G4 TCR and either high or low levels of BTLA were co-cultured with K562 cells pulsed with 50 pM 9V NY-ESO peptide. A 3-fold serial dilution of hBTLA- aProxlM-02 (comprising the BTLA scFv 2.8.6 linked to CD8 VHH21) was prepared, starting at a final concentration of 333 nM, and added at the initiation of co-culture. After 18 h, cells were stained using Zombie Violet viability dye (1 :1000, BioLegend)) and CD69-APC (1 :200; BioLegend) to assess T-cell activation. (B) Jurkat T cell activation and comparison of hBTLA- aProxlM-02, protease-cleaved variant, and dummy control. Jurkat cells expressing the 1 G4 TCR were co-cultured with K562 cells pulsed with 50 pM 9V NY-ESO peptide. A 10-fold serial dilution (starting at 400 nM) was prepared for: (i) hBTLA-aProxlM-02, (ii) 3C-protease-treated hBTLA-aProxlM-02, and (iii) a “dummy” control molecule consisting of the 2.8.6 scFv lacking a CD8-binding domain. After 18 h, cells were stained with Zombie Violet (1 :1000, BioLegend) and CD69-APC (1 :200; BioLegend) and analysed by flow cytometry. Jurkat treatment with intact hBTLA-aProxlM-02 resulted in reduced expression of the CD69 activation marker. SC- cleaved aProxIM and BTLA dummy molecules showed reduced IC50 (~100-fold reduction). Figure 17 shows expanding the kinase-recruitment strategy to the CD2 effector receptors. (A) Bispecific molecules (aProxIM) recruiting CD8 and PD-1 induce phosphorylation of PD-1 via recruitment of the Lek kinase, which binds the intracellular domain (ICD) of CD8. To test whether this strategy can extend to other effector receptors, the inventors generated a chimeric CD8 construct in which the CD8 ICD was replaced with the CD2 ICD. (B) Wild-type Jurkat cells (CD4“CD8“) were stimulated via the TCR using peptide- MHC complexes and treated with aProxlM-12 or aProxlM-13 (open circles / squares). Under these conditions, no changes in NFAT-luciferase reporter activity were observed. In contrast, Jurkat cells expressing the CD8 / CD2 chimaera, comprising the extracellular and transmembrane domains of CD8a fused to the CD2 ICD, showed reduced NFAT-luciferase activity upon aProxlM-12 (CD8xPD-1 binder) treatment (black circles), whereas RAKL-13 (CD4xPD-1 binder) had no effect (black squares).

[0093] Figure 18 shows suppression of T-cell activation by bispecific aProxIM molecules targeting PD-1 and the kinase-associated receptor CD2. Jurkat cells expressing the 1 G4 TCR and an NFAT-Luciferase reporter were co-cultured with K562 cells expressing NY-ESO- 1 peptide presented by HLA-A*02:01. T-cell activation was quantified by luminescence measured 18 h after initiation of co-culture. (A) Jurkat cells were treated with a titration series of bispecific aProxIM molecules containing PD-1 - and CD2-specific scFvs (aProxlM-104, - 106, -131 , -132, -133). Benchmark PD-1 agonist antibodies Peresolimab and Rosnilimab were included for comparison. The aProxlM-1xx series lacks Fc domains. (B) Jurkat cells were treated with CD2-binding (CD58 extracellular domain) x PD-1 (scFv) bispecific molecules aProxlM-701 , -702, and -703. aProxlM-704, composed of CD58 linked to a Palivizumab scFv lacking PD-1 specificity, served as a negative control. (C) Jurkat cells were treated with aProxlM-725, -726, or the negative control aProxlM-704. aProxlM-725 and -726 incorporate engineered CD58 variants modified to reduce glycosylation and improve solubility; aProxIM- 704 binds CD2 but not PD-1. (D) Comparison with benchmark antibodies. Jurkat cells were treated with Siplizumab (anti-CD2), Peresolimab (anti-PD-1 agonist), or Rosnilimab (anti-PD- 1 agonist) as indicated. Jurkat treatment with aProxIMs targeting CD2 and PD-1 , but not control aProxIMs or control antibodies, led to a reduction in NFAT-luciferase reporter activity.

[0094] The patent, scientific and technical literature referred to herein establish knowledge that was available to those skilled in the art at the time of filing. The entire disclosures of the issued patents, published and pending patent applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of any inconsistencies, the present disclosure will prevail. Various aspects of the invention are described in further detail below.

[0095] DETAILED DESCRIPTION

[0096] Multivalent proteins

[0097] Provided herein are multivalent proteins comprising: a. a first region capable of binding to one or more effector proteins, the effector protein comprising or associated with protein kinase activity; and b. a second region capable of binding to one or more target proteins, wherein the target protein signals through a phosphorylation mechanism.

[0098] As used herein, the term “multivalent protein” refers to a multispecific polypeptide comprising two or more regions capable of binding to two or more distinct epitopes. In some examples, multivalent protein may bind one or more epitopes of a first protein and in addition bind one or more epitopes of second protein. For example, the multivalent protein may bind to an effector protein and a target protein. The multivalent protein functions to bring the effector protein and target protein into proximity with one another to initiate a signalling cascade in the absence of target protein ligand. In one embodiment, the multivalent protein is a bivalent protein and comprises two regions capable of binding two distinct epitopes. As outlined in the examples herein, the inventors surprisingly identified that a multivalent protein targeting CD8 (effector protein) and PD-1 (target protein) on the same cell in cis drives PD-1 phosphorylation in the absence of PD-L1 . Without wishing to be bound by theory, the inventors believe that because CD8 is intracellularly associated with Src family kinases, bringing CD8 into proximity with PD- 1 allows the Src family kinases to phosphorylate two intracellular tyrosines on the ITSM and ITIM domains of PD-1 and subsequently induce cellular signalling.

[0099] The multivalent proteins may specifically bind to an effector and target protein. The term "specifically binds" as used herein means the ability of a protein (e.g. multivalent protein), to bind to one or more of its desired binding partners (e.g. effector and / or target protein) such that its affinity or avidity is at least 10 50, 100, 250 or 500 greater than the average affinity or avidity of the same protein to a collection of random peptides or polypeptides of sufficient statistical size. A specifically binding protein need not bind exclusively to a single target molecule but may specifically bind to a non-target molecule due to similarity in structural conformation between the target and non-target (e.g., paralogs or orthologs). Those of skill will recognize that specific binding to a molecule having the same function in a different species of animal (i.e., ortholog) or to a non-target molecule having a substantially similar epitope as the target molecule (e.g., paralog) is possible and does not detract from the specificity of binding which is determined relative to a statistically valid collection of unique non-targets (e.g., random polypeptides). Thus, multivalent proteins as described herein may specifically bind to more than one distinct species of target and / or effector molecule due to cross-reactivity. Generally, such off-target specific binding is mitigated by reducing affinity or avidity for undesired targets. Solid-phase ELISA immunoassays or Biacore measurements can be used to determine specific binding between two proteins.

[0100] Suitably, the multivalent proteins bind to effector and target proteins in cis. That is to say that the effector and target proteins may be present on the same cell. This is in contrast to “trans” binding which would involve binding to a target and effector protein present on two different cells. Without being bound by theory, binding the target and effector protein in the same cell (in cis) may provide for changes in intracellular signalling.

[0101] Extracellular domains and cell surface receptor proteins

[0102] Suitably, the effector protein and / or target protein each comprise one or more extracellular domains. As used herein, the term “extracellular domains” refers to a portion of a protein which is capable of interacting with the extracellular environment. Suitably, an extracellular domain refers to a portion of a transmembrane protein which is sufficient to bind to a ligand or receptor and effectively transmit a signal to a cell. Suitably, an extracellular domain is the entire amino acid sequence of a transmembrane protein which is external of a cell or the cell membrane. Suitably, an extracellular domain is the portion of an amino acid sequence of a transmembrane protein which is external of a cell or the cell membrane and is needed for signal transduction and / or ligand binding as may be assayed using methods known in the art (e.g., in vitro ligand binding and / or cellular activation assays).

[0103] Suitably, an extracellular domain refers to a protein or portion thereof which is capable of interacting with the extracellular environment and is anchored to the cell membrane (e.g. not a transmembrane protein). For example, proteins may be anchored to the cell membrane by any one or more of a lipid anchor (such as a glycosylphosphatidylinositol (GPI) anchor, a Phenyl group anchor, or a fatty acyl group anchor), or a A-kinase anchoring proteins (AKAPs).

[0104] Suitably, extracellular domains may include post-translational modifications such as a covalent modification or enzymatic modification. Examples of post-translation modifications include, but are not limited to, acylation, acetylation, alkylation (including methylation), biotinylation, butyrylation, carbamylation, carbonylation, deamidation, deiminiation, diphthamide formation, disulfide bridge formation, eliminylation, flavin attachment, formylation, gamma-carboxylation, glutamylation, glycylation, glycosylation, glypiation, heme C attachment, hydroxylation, hypusine formation, iodination, isoprenylation, lipidation, lipoylation, malonylation, methylation, myristolylation, oxidation, palmitoylation, pegylation, phosphopantetheinylation, phosphorylation, prenylation, propionylation, retinylidene Schiff base formation, S-glutathionylation, S-nitrosylation, S-sulfenylation, selenation, succinylation, sulfination, ubiquitination, and C-terminal amidation. In some examples, extracellular domains may be glycosylated. Suitably, extracellular domains may include one or more glycopeptide epitopes. “Glycopeptide epitope” refers to an epitope that includes both part of a peptide sequence and at least part of a glycan.

[0105] Preferably, the effector protein and / or target protein each comprise one or more cell surface receptor proteins. As used herein, the term “cell surface receptor proteins” refers to cell membrane associated proteins which perform signal transduction, in which an extracellular signal is converted into an intercellular signal. Suitably, cell surface receptor proteins may be cell surface, membrane-anchored, or integral proteins that bind to extracellular ligand molecules. Suitably, the cell surface receptor proteins are transmembrane proteins embedded in the plasma membrane of target cells comprising at least an extracellular domain containing a ligand-binding site, a transmembrane domain, and an intracellular domain that transmits a signal inside the cell. Suitably, cell surface receptor proteins may be specific to individual cell types. Non-limiting examples of cell surface receptor proteins include ion channel linked receptors, enzyme-linked receptors, G protein-coupled receptors, adrenergic receptor, olfactory receptors, receptor tyrosine kinases, epidermal growth factor receptor, insulin receptor, fibroblast growth factor receptors, high affinity neurotrophin receptors, ephrin receptors, integrins, low affinity nerve growth factor receptor, NMDA receptor, and immune receptors. For example, PD-1 , BTLA, CD5, CD6, CD200R, VISTA, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, CD66, LAIR1 , ILT-2, ILT-4, CD31 , CD4, CD8, CD48 CD2, CD59, CD55, CD24, and CD14. For example, PD-1 , BTLA, CD200R, CD4 and CD8.

[0106] Effector proteins

[0107] Suitably, at least one region of the multivalent protein is capable of binding to one or more effector proteins. Suitably, the effector protein comprises or is associated with protein kinase activity. Suitably, the effector protein comprises protein kinase activity. Suitably, the effector protein is associated with protein kinase activity.

[0108] As used herein, the term “effector protein” refers to a protein that is either directly or indirectly involved in the phosphorylation of a target protein. Therefore, the target protein may be directly phosphorylated by the effector protein (e.g. the effector protein comprises protein kinase activity). As used herein, the term “protein kinase activity” refers to the ability to selectively modify a protein by phosphorylation. Alternatively, the target protein may be indirectly phosphorylated due to the effector protein being associated with protein kinase activity. As used herein, the term “associated with protein kinase activity” refers to a protein having the ability to interact (e.g. bind to and / or recruit) proteins with kinase activity. Effector proteins associated with protein kinase activity typically do not have intrinsic kinase activity but bind to proteins which comprise kinase activity. For examples, cell surface receptors CD2, CD4, CD8, CD14, CD24, CD48, CD55, and CD59 are understood to be associated with protein kinase activity because they intracellularly interact with Src family kinases. Suitably, the effector protein is selected from CD2, CD4, CD8, CD14, CD24, CD48, CD55, and / or CD59. Suitably, the effector protein is selected from CD4 and / or CD8.

[0109] As used herein, the term “phosphorylation” refers to the addition of a phosphate group to a molecule. Suitably, the phosphate group may replace a side chain hydroxyl group of an amino acid. Accordingly, a molecule that is “phosphorylated” is one that comprises a phosphate group. Suitably, the molecule is a protein, for example an amino acid residue of a protein. In vitro, phosphorylation is a common reversible post-translational modification of one or more amino acid residues of a protein, wherein the one or more amino acid residue side chain hydroxyl group has been substituted for a phosphate group.

[0110] In vivo, phosphorylation is commonly associated with the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways. Signal transduction pathways are characterised by one or more proteins physically sensing cues, either through ligand binding (e.g. PD-L1 binding PD-1), cleavage or some other response, that then relay the signal to second messengers and signalling enzymes. In the case of phosphorylation, these receptors activate downstream kinases, which then phosphorylate and activate their cognate downstream substrates, including additional kinases, until the specific response is achieved. Accordingly, it may be favourable to regulate the levels of phosphorylation of proteins to control said signal transduction pathways.

[0111] Phosphorylation is typically catalysed by kinases. The most commonly phosphorylated amino acid residues are tyrosine, serine, threonine, and histidine; however, other amino acids can also be phosphorylated, including arginine, lysine, aspartic acid, glutamic acid and cysteine. Typically, protein kinases are named according to the residues they phosphorylate. For example, a protein which phosphorylates tyrosine residues is termed a “protein tyrosine kinase”.

[0112] Kinases are enzymes that facilitate phosphate group transfer to substrates. ATP is the cosubstrate for almost all protein kinases, although guanosine triphosphate is used by a small number of kinases.

[0113] Suitably, the effector protein comprises a receptor-protein tyrosine kinase. Suitably, the effector protein is a cell surface receptor that interacts with one or more intracellular protein kinases. Receptor-protein tyrosine kinase

[0114] Suitably, the effector protein comprises a receptor-protein tyrosine kinase (RTK).

[0115] As used herein, the term “receptor-protein tyrosine kinase” refers to one class of protein kinases which comprise of transmembrane receptors and are characterised by their cytoplasmic regions' intrinsic tyrosine kinase activity.

[0116] Receptor-protein tyrosine kinases play a critical role in regulating cell differentiation, proliferation, survival, metabolism, and migration by phosphorylating tyrosine residues of proteins. There are 58 unique known receptor-protein tyrosine kinases. All have an extracellular region with ligand-binding domains, a single transmembrane alpha-helix, and a cytoplasmic region consisting of a juxta-membrane domain, a tyrosine kinase domain, and a C-terminal tail. The intracellular signalling pathways associated with receptor- protein tyrosine kinases are triggered when protein kinases bind to phosphotyrosines on the receptor-protein tyrosine kinase and subsequently become phosphorylated.

[0117] There are approximately 20 different RTK classes. Suitably, the receptor-protein tyrosine kinase may be in the RTK class I (EGF receptor family) (ErbB family), RTK class II (Insulin receptor family), RTK class III (PDGF receptor family), RTK class IV (VEGF receptors family), RTK class V (FGF receptor family), RTK class VI (CCK receptor family), RTK class VII (NGF receptor family), RTK class VIII (HGF receptor family), RTK class IX (Eph receptor family), RTK class X (AXL receptor family), RTK class XI (TIE receptor family), RTK class XII (RYK receptor family), RTK class XIII (DDR receptor family), RTK class XIV (RET receptor family), RTK class XV (ROS receptor family), RTK class XVI (LTK receptor family), RTK class XVII (ROR receptor family), RTK class XVIII (MuSK receptor family), RTK class XIX (LMR receptor), or RTK class XX (Undetermined). The 58 known receptor- protein tyrosine kinases include EGFR, ErbB2, ErbB3, ErbB4, InsR, IGF1 R, InsRR, PDGFRa, PDGFRp, CSF1 R / Fms, Kit / SCFR, Flt3 / Flk2, VEGFR1 / Flt1 , VEGFR2 / KDR, VEGFR3 / Flt4, FGFR1 , FGFR2, FGFR3, FGFR4, PTK7 / CCK4, TrkA, TrkB, TrkC, Ror1 , Ror2, MuSK, Met, Ron, Axl, Mer, Tyro3, Tie1 , Tie2, EphA1 -8, EphAIO, EphB1-4, EphB6, Ret, Ryk, DDR1 , DDR2, Ros, LMR1 , LMR2, LMR3, ALK, LTK, and SuRTK106 / STYK1 .

[0118] Cell surface receptor that interacts with one or more intracellular protein kinases Suitably, the effector protein comprises a cell surface receptor that interacts with one or more intracellular protein kinases. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is selected from CD2, CD4, CD8, CD14, CD24, CD48, CD55, and CD59. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD2. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD4. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD8. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD14. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD24. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD48. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD55. Suitably, the cell surface receptor that interacts with one or more intracellular protein kinases is CD59.

[0119] CD2 is a cell surface glycoprotein of T cells. CD2 interacts with lymphocyte function- associated antigen CD58 (LFA-3) and CD48 / BCM1 to mediate adhesion between T-cells and other cell types. CD2 is implicated in the triggering of T-cells and the cytoplasmic domain is implicated in the signalling function.

[0120] CD2’s natural ligand, CD58 (also known as LFA-3), is widely expressed on antigen-presenting cells and other hematopoietic and non-hematopoietic cell types. CD2 and CD58 form a well- characterized receptor-ligand pairwith moderate to high affinity (typically in the low micromolar range), mediating stable intercellular adhesion and contributing to immunological synapse formation. The interaction interface is structurally defined and highly specific, allowing the extracellular domain of CD58 to bind CD2 effectively without requiring antibody-derived targeting domains. Accordingly, CD58 can be employed as a natural and efficient CD2-binding module in place of anti-CD2 antibodies or scFvs such as Siplizumab or RPA-2.10. This enables CD58-based constructs to selectively engage CD2 while avoiding the sequence liabilities or epitope constraints associated with antibody binders.

[0121] CD2 contains an intracellular domain that associates with Src family kinases, including Lek and Fyn, which contribute to phosphorylation-dependent signalling events upon receptor engagement. The binding of CD58 to CD2 promotes receptor clustering at the cell surface, thereby increasing the local concentration of CD2-associated kinases on the intracellular side of the membrane. Incorporation of the CD58 extracellular domain into the constructs described herein has been shown to facilitate the selective recruitment of CD2 in cis, on the same cell, bringing CD2-associated kinases into proximity with a target inhibitory receptor such as PD-1 . This natural ligand-mediated recruitment provides a mechanism by which CD58-containing aProxIM constructs can induce phosphorylation of the targeted receptor, functioning as an effective kinase-recruitment module within the platform.

[0122] CD4 is a cell surface glycoprotein of T cells. CD4 is a co-receptor of the T cell receptor (TCR) and assists the latter in communicating with antigen-presenting cells. CD4 plays an essential role in the immune response and serves multiple functions in responses against both external and internal offenses. In T-cells, CD4 functions primarily as a coreceptor for MHC class II molecule:peptide complex. CD4 interacts simultaneously with the T-cell receptor (TCR) and the MHC class II presented by antigen presenting cells (APCs). In turn, recruits the Src kinase Lek to the vicinity of the TCR-CD3 complex. Lek then initiates different intracellular signalling pathways by phosphorylating various substrates ultimately leading to lymphokine production, motility, adhesion and activation of T-helper cells.

[0123] CD8 is a cell surface glycoprotein of cytotoxic T cells. CD8 mediates efficient cell-cell interactions within the immune system. CD8 acts as a coreceptor with the T-cell receptor on the T lymphocyte to recognise antigens displayed by an antigen presenting cell in the context of class I MHC molecules. Lek then initiates different intracellular signalling pathways by phosphorylating various substrates ultimately leading to lymphokine production, motility, adhesion and activation of cytotoxic T-lymphocytes (CTLs). This mechanism enables CTLs to recognize and eliminate infected cells and tumour cells. In NK-cells, the presence of CD8 homodimers at the cell surface provides a survival mechanism allowing conjugation and lysis of multiple target cells. CD8 homodimer molecules also promote the survival and differentiation of activated lymphocytes into memory CD8 T-cells.

[0124] CD14 is a cell surface glycoprotein preferentially expressed on monocytes / macrophages. It cooperates with other proteins to mediate the innate immune response to bacterial lipopolysaccharide, and to viruses. CD14 acts via MyD88, TIRAP and TRAF6, leading to NF- kappa-B activation, cytokine secretion and the inflammatory response. Acts as a coreceptor for TLR2:TLR6 heterodimer in response to diacylated lipopeptides and for TLR2:TLR1 heterodimer in response to tri-acylated lipopeptides, these clusters trigger signalling from the cell surface and subsequently are targeted to the Golgi in a lipid-raft dependent pathway.

[0125] CD24 is a sialoglycoprotein that is expressed on mature granulocytes and B cells and modulates growth and differentiation signals to these cells. CD24 have a pivotal role in cell differentiation of different cell types. Signalling via CD24 may be triggered by the binding of a lectin-like ligand to the CD24 carbohydrates and transduces by the release of second messengers derived from the GPI-anchor. CD24 is believed to modulate B-cell activation responses, promotes AG-dependent proliferation of B-cells and prevents their terminal differentiation into antibody-forming cells. In association with SIGLEC10 CD24 may be involved in the selective suppression of the immune response to danger-associated molecular patterns (DAMPs) such as HMGB1 , HSP70 and HSP90. CD24 is believed to play a role in the control of autoimmunity.

[0126] CD48 is a cell surface glycosylphosphatidylinositol (GPI)-anchored cell surface glycoprotein that interacts via its N-terminal immunoglobulin domain with cell surface receptors including 2B4 / CD244 or CD2 to regulate immune cell function and activation. CD48 participates in T- cell signalling transduction by associating with CD2 and efficiently bringing the Src family protein kinase Lek and Lat to the TCR-CD3 complex. In turn, promotes LCK phosphorylation and subsequent activation. Induces the phosphorylation of the cytoplasmic immunoreceptor tyrosine switch motifs (ITSMs) of CD244 initiating a series of signalling events that leads to the generation of the immunological synapse and the directed release of cytolytic granules containing perforin and granzymes by T-lymphocytes and NK-cells.

[0127] CD55 is a glycoprotein involved in the regulation of the complement cascade. Binding of CD55 to complement proteins accelerates their decay, thereby disrupting the cascade and preventing damage to host cells. CD55 is understood to inhibit complement activation by destabilising and preventing the formation of C3 and C5 convertases, which prevents complement damage.

[0128] CD59 is a cell surface glycoprotein that regulates complement-mediated cell lysis, and it is involved in lymphocyte signal transduction. As CD59 can be complexed to a protein tyrosine kinase, it also plays a role in signal transduction pathways in the activation of T cells.

[0129] As used herein, the term “intracellular protein kinases” refers to protein kinases situated within the intracellular portion of the cell which phosphorylate residues on proteins.

[0130] Suitably, the one or more intracellular protein kinases comprises one or more Src family kinases. The Src kinase family is a family of membrane-associated non-receptor tyrosine kinases which function to transduce signals related to cellular processes such as proliferation, differentiation, motility, and adhesion through phosphorylating tyrosine residues on target proteins.

[0131] The Src family kinases includes two sub families: SrcA and SrcB. Suitably, the one or more Src family kinases may be selected from sub families SrcA or SrcB. The SrcA sub family includes Src, Yes, Fyn, and Fgr. Suitably, the one or more Src family kinases may be selected from Src, Yes, Fyn, and / or Fgr. The SrcB sub family includes Lek, Hck, Blk, and Lyn. Suitably, the one or more Src family kinases may be selected from Lek, Hck, Blk, and / or Lyn. Suitably, the one or more Src family kinases may be selected from Src, Yes, Fyn, Fgr, Lek, Hck, Blk, and / or Lyn. Suitably, the Src family kinase is a Src related kinase, such as the Yes-related kinase (Yrk).

[0132] Src family kinases contain six conserved domains: a N-terminal myristoylated segment, a SH2 domain, a SH3 domain, a linker region, a tyrosine kinase domain, and C-terminal tail.

[0133] The Src family kinase Lek (lymphocyte-specific protein tyrosine kinase) is a protein which localises to the plasma membrane of cells and pericentrosomal vesicles. Lek contains N- terminal sites for myristylation and palmitylation, a tyrosine kinase domain, and SH2 and SH3 domains which are involved in mediating protein-protein interactions with phosphotyrosine- containing and proline-rich motifs, respectively. Lek plays an essential role in the selection and maturation of developing T-cells in the thymus and in the function of mature T-cells. Lek plays a key role in T-cell antigen receptor (TCR)-linked signal transduction pathways. Lek is constitutively associated with the cytoplasmic portions of the CD4 and CD8 surface receptors. Association of the TCR with a peptide antigen-bound MHC complex facilitates the interaction of CD4 and CD8 with MHC class II and class I molecules, respectively, thereby recruiting the associated Lek protein to the vicinity of the TCR / CD3 complex. Lek then phosphorylates tyrosine residues within the immunoreceptor tyrosine-based activation motifs (ITAM) of the cytoplasmic tails of the TCR-gamma chains and CD3 subunits, initiating the TCR / CD3 signalling pathway. Once stimulated, the TCR recruits the tyrosine kinase ZAP70, that becomes phosphorylated and activated by Lek. Following this, a large number of signalling molecules are recruited, ultimately leading to lymphokine production. Lek also contributes to signalling by other receptor molecules. Associates directly with the cytoplasmic tail of CD2, which leads to hyperphosphorylation and activation of Lek. Also plays a role in the IL2 receptor-linked signalling pathway that controls the T-cell proliferative response. Binding of IL2 to its receptor results in increased activity of Lek. Is expressed at all stages of thymocyte development and is required for the regulation of maturation events that are governed by both pre-TCR and mature alpha beta TCR. Phosphorylates other substrates including RUNX3, PTK2B / PYK2, the microtubule-associated protein MAPT, RHOH or TYROBP. Interacts with FYB2.

[0134] As outlined in the examples herein, the inventors surprisingly identified that following treatment of HEK293 cells expressing CD8, PD-1 and Lek, PD-1 pTyr was noticeably increased after treatment with the multivalent proteins of the invention. When Lek was not expressed in HEK293 cells, there was no phosphorylation of PD-1 .

[0135] FGR proto-oncogene, Src family tyrosine kinase (Fgr) contains N-terminal sites for myristylation and palmitylation, a PTK domain, and SH2 and SH3 domains which are involved in mediating protein-protein interactions with phosphotyrosine-containing and proline-rich motifs, respectively. The protein localizes to plasma membrane ruffles, and functions as a negative regulator of cell migration and adhesion triggered by the beta-2 integrin signal transduction pathway. Fgr kinase: regulates immune responses, including the functions of neutrophils, monocytes, macrophages, and mast cells; regulates cytoskeleton remodeling in response to extracellular stimuli; regulates phagocytosis; contributes to hemorrhage-induced thalamic pain by activating NF-kappaB / ERK1 / 2 pathways; is required for proinflammatory macrophage activation during diet-induced obesity. Fgr kinase is involved in many diseases, including: Hematologic cancer, Placental insufficiency, and Sepsis-associated encephalopathy (SAE).Yes Kinase is a non-receptor protein tyrosine kinase that is involved in the regulation of cell growth and survival, apoptosis, cell-cell adhesion, cytoskeleton remodelling, and differentiation. Stimulation by receptor tyrosine kinases (RTKs) including EGFR, PDGFR, CSF1 R and FGFR leads to recruitment of YES1 to the phosphorylated receptor, and activation and phosphorylation of downstream substrates. Upon EGFR activation, Yes promotes the phosphorylation of PARD3 to favour epithelial tight junction assembly. Yes participates in the phosphorylation of specific junctional components such as CTNND1 by stimulating the FYN and FER tyrosine kinases at cell-cell contacts. Upon T-cell stimulation by CXCL12, Yes phosphorylates collapsin response mediator protein 2 / DPYSL2 and induces T-cell migration. Yes participates in CD95L / FASLG signalling pathway and mediates AKT-mediated cell migration. Yes plays a role in cell cycle progression by phosphorylating the cyclin-dependent kinase 4 / CDK4 thus regulating the G1 phase and is also involved in G2 / M progression and cytokinesis.

[0136] Fyn is a non-receptor tyrosine-protein kinase that plays a role in many biological processes including regulation of cell growth and survival, cell adhesion, integrin-mediated signalling, cytoskeletal remodelling, cell motility, immune response and axon guidance . Inactive Fyn is phosphorylated on its C-terminal tail within the catalytic domain. Following activation by PKA, the protein subsequently associates with PTK2 / FAK1 , allowing PTK2 / FAK1 phosphorylation, activation and targeting to focal adhesions. Fyn: is involved in the regulation of cell adhesion and motility through phosphorylation of CTNNBI (beta-catenin) and CTNND1 (delta-catenin); regulates cytoskeletal remodelling by phosphorylating several proteins including the actin regulator WAS and the microtubule-associated proteins MAP2 and MAPT; promotes cell survival by phosphorylating AGAP2 / PIKE-A and preventing its apoptotic cleavage; participates in signal transduction pathways that regulate the integrity of the glomerular slit diaphragm (an essential part of the glomerular filter of the kidney) by phosphorylating several slit diaphragm components including NPHS1 , KIRREL1 and TRPC6; plays a role in neural processes by phosphorylating DPYSL2, a multifunctional adapter protein within the central nervous system, ARHGAP32, a regulator for Rho family GTPases implicated in various neural functions, and SNCA, a small pre-synaptic protein; involved in reelin signalling by mediating phosphorylation of DAB1 following reelin (RELN)-binding to its receptor; participates in the downstream signalling pathways that lead to T-cell differentiation and proliferation following T-cell receptor (TCR) stimulation; phosphorylates PTK2B / PYK2 in response to T-cell receptor activation; and participates in negative feedback regulation of TCR signalling through phosphorylation of PAG1 , thereby promoting interaction between PAG1 and CSK and recruitment of CSK to lipid rafts. Src is a non-receptor protein tyrosine kinase which is activated following engagement of many different classes of cellular receptors including immune response receptors, integrins and other adhesion receptors, receptor protein tyrosine kinases, G protein-coupled receptors as well as cytokine receptors. Src participates in signalling pathways that control a diverse spectrum of biological activities including gene transcription, immune response, cell adhesion, cell cycle progression, apoptosis, migration, and transformation. SRC appears to be one of the primary kinases activated following engagement of receptors and plays a role in the activation of other protein tyrosine kinase (PTK) families. Receptor clustering or dimerization leads to recruitment of SRC to the receptor complexes where it phosphorylates the tyrosine residues within the receptor cytoplasmic domains. Src plays an important role in the regulation of cytoskeletal organization through phosphorylation of specific substrates such as AFAP1. Phosphorylation of AFAP1 allows the SRC SH2 domain to bind AFAP1 and to localize to actin filaments. Cytoskeletal reorganization is also controlled through the phosphorylation of cortactin (CTTN). When cells adhere via focal adhesions to the extracellular matrix, signals are transmitted by integrins into the cell resulting in tyrosine phosphorylation of a number of focal adhesion proteins, including PTK2 / FAK1 and paxillin. In addition to phosphorylating focal adhesion proteins, SRC is also active at the sites of cell-cell contact adherens junctions and phosphorylates substrates such as beta-catenin (CTNNB1), delta-catenin (CTNND1), and plakoglobin (JUP). Anothertype of cell-cell junction, the gap junction, is also a target for SRC, which phosphorylates connexin-43 (GJA1). SRC is implicated in regulation of pre-mRNA-processing and phosphorylates RNA-binding proteins such as KHDRBS1 (Probable). Phosphorylates PKP3 at 'Tyr-195' in response to reactive oxygen species, which may cause the release of PKP3 from desmosome cell junctions into the cytoplasm. Src also plays a role in PDGF-mediated tyrosine phosphorylation of both STAT1 and STAT3, leading to increased DNA binding activity of these transcription factors. Src is also: involved in the RAS pathway through phosphorylation of RASA1 and RASGRF1 ; plays a role in EGF-mediated calcium-activated chloride channel activation; required for epidermal growth factor receptor (EGFR) internalization through phosphorylation of clathrin heavy chain (CLTC and CLTCL1) at 'Tyr-1477'; involved in beta-arrestin (ARRB1 and ARRB2) desensitization through phosphorylation and activation of GRK2, leading to beta-arrestin phosphorylation and internalization; has a critical role in the stimulation of the CDK20 / MAPK3 mitogen-activated protein kinase cascade by epidermal growth factor; plays an important role in osteoclastic bone resorption in conjunction with PTK2B / PYK2; recruited to activated integrins by PTK2B / PYK2, thereby phosphorylating CBL, which in turn induces the activation and recruitment of phosphatidylinositol 3-kinase to the cell membrane in a signalling pathway that is critical for osteoclast function; promotes energy production in osteoclasts by activating mitochondrial cytochrome C oxidase; phosphorylates DDR2 on tyrosine residues, thereby promoting its subsequent autophosphorylation; phosphorylates RUNX3 and COX2 on tyrosine residues, TNK2 on 'Tyr-284' and CBL on 'Tyr-731 '; enhances RIGI-elicited antiviral signalling; phosphorylates PDPK1 at 'Tyr-9', 'Tyr-373' and 'Tyr-376'; phosphorylates BCAR1 at 'Tyr-128'; phosphorylates CBLC at multiple tyrosine residues, phosphorylation at 'Tyr-341 ' activates CBLC E3 activity; phosphorylates synaptic vesicle protein synaptophysin (SYP); involved in anchorage-independent cell growth; required for podosome formation; mediates IL6 signalling by activating YAP1-NOTCH pathway to induce inflammation-induced epithelial regeneration; phosphorylates OTUB1 , promoting deubiquitination of RPTOR; phosphorylates caspase CASP8 at 'Tyr-380' which negatively regulates CASP8 processing and activation, downregulating CASP8 proapoptotic function.

[0137] Hck is a non-receptor tyrosine-protein kinase found in hematopoietic cells that transmits signals from cell surface receptors and plays an important role in the regulation of innate immune responses, including neutrophil, monocyte, macrophage and mast cell functions, phagocytosis, cell survival and proliferation, cell adhesion and migration. HCK acts downstream of receptors that bind the Fc region of immunoglobulins, such as FCGR1A and FCGR2A, but also CSF3R, PLAUR, the receptors for IFNG, IL2, IL6 and IL8, and integrins, such as ITGB1 and ITGB2. During the phagocytic process, Hck mediates mobilization of secretory lysosomes, degranulation, and activation of NADPH oxidase to bring about the respiratory burst. Hck plays a role in the release of inflammatory molecules and promotes reorganization of the actin cytoskeleton and actin polymerization, formation of podosomes and cell protrusions. Hck also inhibits TP73-mediated transcription activation and TP73-mediated apoptosis. Hck also phosphorylates CBL in response to activation of immunoglobulin gamma Fc region receptors and phosphorylates ADAM15, BCR, ELMO1 , FCGR2A, GAB1 , GAB2, RAPGEF1 , STAT5B, TP73, VAV1 and WAS.

[0138] Blk is a non-receptor tyrosine kinase involved in B-lymphocyte development, differentiation and signalling (By similarity). B-cell receptor (BCR) signalling requires a tight regulation of several protein tyrosine kinases and phosphatases, and associated coreceptors. Binding of antigen to the B-cell antigen receptor (BCR) triggers signalling that ultimately leads to B-cell activation. Signaling through BLK plays an important role in transmitting signals through surface immunoglobulins and supports the pro-B to pre-B transition, as well as the signalling for growth arrest and apoptosis downstream of B-cell receptor. Blk specifically binds and phosphorylates CD79A at 'Tyr-188'and 'Tyr-199', as well as CD79B at 'Tyr-196' and 'Tyr-207'. Blk also phosphorylates the immunoglobulin G receptors FCGR2A, FCGR2B and FCGR2C. With FYN and LYN, Blk plays an essential role in pre-B-cell receptor (pre-BCR)-mediated NF- kappa-B activation. Blk also contributes to BTK activation by indirectly stimulating BTK intramolecular autophosphorylation. In pancreatic islets, Blk acts as a modulator of beta-cells function through the up-regulation of PDX1 and NKX6-1 and consequent stimulation of insulin secretion in response to glucose. Target proteins

[0139] Suitably, at least one region of the multivalent protein is capable of binding to one or more target proteins. Suitably, the target protein signals through a phosphorylation mechanism. As used herein, the term “phosphorylation mechanism" in the context of signalling refers to the act of phosphorylation and dephosphorylation for mediating cellular transduction cascades.

[0140] Suitably, the target protein mediates signalling through a tyrosine motif. As used herein, the term “tyrosine motif’ refers to a conserved intracellular sequence of amino acids comprising tyrosine(s) which function to mediate intracellular signalling through a phosphorylation mechanism. When the tyrosine(s) of the tyrosine motifs are phosphorylated upon receptor activation by cognate ligand binding, they serve as docking sites to recruit downstream signalling proteins containing the Src Homology 2 (SH2) domain. Examples of tyrosine motifs are immunoreceptor tyrosine-based activation motif (ITAM), immunoreceptor tyrosine-based inhibitory motif (ITIM), and immunoreceptor tyrosine-based switch motif (ITSM).

[0141] Suitably, the tyrosine motif is selected from one or more of an ITAM, ITSM, or ITIM or a related intracellular motif.

[0142] Suitably, the tyrosine motif is ITAM. ITAMs are characterised by the consensus sequence of YxxL / l-(x)6-8-YxxL / l, where x is any amino acid. Non-limiting examples of target proteins which comprise an ITAM, include CD22, Siglec-14, and CD31.

[0143] Suitably, the tyrosine motif is ITSM. ITSMs are characterised by the consensus sequence of TxYxxV / l, where x is any amino acid. Non-limiting examples of target proteins which comprise an ITSM include PD-1 and BTLA.

[0144] Suitably, the tyrosine motif is ITIM. ITIMs are characterised by the consensus sequence S / l / V / LxYxxLA / , where x is any amino acid. Non-limiting examples of target proteins which comprise an ITIM include PD-1 , FcyRIIB, BTLA, TIGIT, CD22, Siglec-7, Siglec-9, Siglec-6, CD300a, CD72, LAIR1 , ILT-2, and ILT-4.

[0145] Suitably, the tyrosine is an intracellular motif related to ITAM, ITSM, or ITIM. Non-limiting examples of target proteins comprising related intracellular motifs include CD200R1 (YxxYxxxxxxY, where x is any amino acid), CD5 (ITAM-like), TIM-3 (comprises five tyrosines), Siglec-6 (ITIM-like), and CTLA-4 (YVKM).

[0146] Suitably the target protein is an immune checkpoint receptor. Suitably the target protein is a cytokine receptor. Suitably the target protein is a growth factor receptor. Suitably the target protein is an interferon receptor. Suitably the target protein is a tumour necrosis factor receptor. Suitably, the target protein comprises an inhibitory receptor protein.

[0147] As used herein, “immune checkpoints” are pathways that modulate the immune response and can be either stimulatory or inhibitory. Stimulatory immune checkpoint pathways function to increase the immune response by promoting the activation and proliferation of immune cells. In contrast, inhibitory immune checkpoint pathways function to decrease the immune response by preventing immune cell activation to avoid excessive inflammation and autoimmune attack. Or in other words, inhibitory immune checkpoint pathways have an immunosuppressive action.

[0148] The term “immune checkpoint receptor” as used herein, refers to a receptor of an immune checkpoint pathway which is bound by a cognate ligand in order to stimulate or inhibit an immune response. Typically, the immune checkpoint receptors are expressed on the cell surface.

[0149] As used herein, the term “inhibitory receptor protein” refers to a receptor protein which is typically bound by a ligand in order to produce an immunosuppressive response.

[0150] Non-limiting examples of inhibitory receptor proteins include PD-1 , BTLA, CD5, CD6, CD200R, VISTA, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, CD66, LAIR1 , ILT-2, ILT-4, CD31 , SIRPa, members of the CEACAM family, members of the SLAM family of surface receptors, family of surface receptors and members of the KIR family of surface receptors.

[0151] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is selected from PD-1 , BTLA, CD5, CD6, CD200R, VISTA, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, CD66, LAIR1 , ILT-2, ILT-4, CD31 , SIRPa, members of the CEACAM family, members of the SLAM family of surface receptors, family of surface receptors and members of the KIR family of surface receptors.

[0152] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is selected from PD-1 , BTLA, and / or CD200R1. Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is selected from PD-1 , and / or BTLA. Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is selected from PD-1 , and / or CD200R1 . Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is selected from BTLA, and / or CD200R1 .

[0153] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is PD-1. Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is BTLA. Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CD200R1 .

[0154] PD-1 (also known in the state of the art as PD1 , CD279, PDCD1 , SLEB2, hPD-1 , hPD-l, hSLE1 , and Programmed cell death 1) is a type I transmembrane protein containing an Ig Variable-type (V-type) amino-terminal extracellular domain, a transmembrane region, and a cytoplasmic tail with an immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoreceptor tyrosine-based switch motif (ITSM). Homo sapiens PD-1 has the amino acid sequence as shown in SEQ ID NO. 128. Mus musculus PD-1 has the amino acid sequence as shown in SEQ ID NO. 129. PD-1 is an inhibitory receptor protein involved in both adaptive and innate immune responses, and is expressed on activated T cells, natural killer (NK) cells and B lymphocytes, macrophages, dendritic cells (DCs) and monocytes. PD-1 is involved in suppressing the immune system during particular events such as pregnancy, tissue allographs, autoimmune disease and other disease states such as hepatitis and cancer. Ligands of PD-1 are Programmed death-ligand 1 and 2 (PD-L1 and PD-L2). The formation of the PD-1 / PD-L1 (or PD-L2) complex transmits an inhibitory signal which results in reduction of the proliferation of PD-1 positive T cells, inhibits their cytokine secretion and induces apoptosis. In autoimmune diseases, abnormal immune responses arise to self-antigens. The PD-1 pathway, as an inhibitory signal, controls the induction and maintenance of tolerance to self-antigens in the context of autoimmunity. Therefore, the PD-1 pathway plays a significant role in autoimmune disorders, including inflammatory bowel disease (IBD), systemic lupus erythematosus (SLE), Type 1 diabetes (T1 D), systemic vasculitis, myositis, autoimmune encephalomyelitis, autoimmune hepatitis, Behcet’s disease, myasthenia gravis, autoimmune uveitis, Sjogren’s syndrome, and ankylosing spondylitis.

[0155] Homo sapiens PD-1 amino acid sequence - SEQ ID NO. 128:

[0156] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNT SESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDS GTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGGLLGS LVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTPEPPVPC VPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

[0157] Mus musculus PD-1 amino acid sequence - SEQ ID NO. 129:

[0158] MWVRQVPWSFTWAVLQLSWQSGWLLEVPNGPWRSLTFYPAWLTVSEGANATFTCSLSN WSEDLMLNWNRLSPSNQTEKQAAFCNGLSQPVQDARFQIIQLPNRHDFHMNILDTRRNDS GIYLCGAISLHPKAKIEESPGAELVVTERILETSTRYPSPSPKPEGRFQGMVIGIMSALVGIPV LLLLAWALAVFCSTSMSEARGAGSKDDTLKEEPSAAPVPSVAYEELDFQGREKTPELPTAC VHTEYATIVFTEGLGASAMGRRGSADGLQGPRPPRHEDGHCSWPL BTLA (also known in the state of the art as BTLA1 , CD272, and B and T lymphocyte associated) is a type I transmembrane glycoprotein containing an extracellular domain, a transmembrane domain and a cytoplasmic domain a transmembrane region, and a cytoplasmic domain containing the growth factor receptor-bound protein-2 (Grb-2) recognition motif, the immunoreceptor tyrosine-based inhibitory motif (ITIM), and the immunoreceptor tyrosine-based switch motif (ITSM). BTLA has the amino acid sequence as shown in SEQ ID NO. 130. BTLA is an inhibitory receptor protein found on various immune cells, including T cells, B cells, and dendritic cells. BTLA is involved in numerous physio pathological processes, such as cancer, inflammatory diseases, autoimmune diseases, infectious diseases, and transplantation rejection. HVEM binding activates tyrosine phosphorylation of the ITIM in BTLA and leads to the recruitment of the Src homology domain 2 (SH2)-containing protein tyrosine phosphatases, SHP-1 and SHP-2, which generally mediate immunosuppressive effects.

[0159] Homo sapiens BTLA amino acid sequence - SEQ ID NO. 130:

[0160] MKTLPAMLGTGKLFWVFFLIPYLDIWNIHGKESCDVQLYIKRQSEHSILAGDPFELECPVKYC ANRPHVTWCKLNGTTCVKLEDRQTSWKEEKNISFFILHFEPVLPNDNGSYRCSANFQSNLI ESHSTTLYVTDVKSASERPSKDEMASRPWLLYRLLPLGGLPLLITTCFCLFCCLRRHQGKQ NELSDTAGREINLVDAHLKSEQTEASTRQNSQVLLSETGIYDNDPDLCFRMQEGSEVYSNP CLEENKPGIVYASLNHSVIGPNSRLARNVKEAPTEYASICVRS

[0161] CD200R1 (also known in the state of the art as CD200R, HCRTR2, MOX2R, OX2R, and CD200 receptor 1) is a membrane glycoprotein receptor which is expressed on the surface of myeloid cells and CD4+ T cells. Homo sapiens CD2000R1 has the amino acid sequence as shown in SEQ ID NO. 131. CD2000R1 limits inflammation by inhibiting the expression of pro- inflammatory molecules including TNF-alpha, interferons, and inducible nitric oxide synthase (iNOS) in response to selected stimuli.

[0162] Homo sapiens CD200R1 amino acid sequence - SEQ ID NO. 131 :

[0163] MLCPWRTANLGLLLILTIFLVAEAEGAAQPNNSLMLQTSKENHALASSSLCMDEKQITQNYS KVLAEVNTSWPVKMATNAVLCCPPIALRNLIIITWEIILRGQPSCTKAYKKETNETKETNCTDE RITWVSRPDQNSDLQIRTVAITHDGYYRCIMVTPDGNFHRGYHLQVLVTPEVTLFQNRNRT AVCKAVAGKPAAHISWIPEGDCATKQEYWSNGTVTVKSTCHWEVHNVSTVTCHVSHLTGN KSLYIELLPVPGAKKSAKLYIPYIILTIIILTIVGFIWLLKVNGCRKYKLNKTESTPVVEEDEMQP YASYTEKNNPLYDTTNKVKASEALQSEVDTDLHTL Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CD5. CD5 is also known in the state of the art as LEU1 , T1 , and CD5 molecule.

[0164] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is TIM-3. TIM-3 is also known in the state of the art as HAVCR2, HAVcr-2, KIM-3, TIM3, TIMD-3, TIMD3, Tim-3, CD366, hepatitis A virus cellular receptor 2, and SPTCL.

[0165] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is TIGIT. TIGIT is also known in the state of the art as VSIG9, VSTM3, WUCAM, T-cell immunoreceptor with Ig and ITIM domains, and T cell immunoreceptor with Ig and ITIM domains.

[0166] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CD22. CD22 is also known in the state of the art as SIGLEC-2, SIGLEC2, and CD22 molecule.

[0167] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is Siglec-7. Siglec-7 is also known in the state of the art as SIGLEC7, AIRM1 , CD328, CDw328, D-siglec, QA79, SIGLEC-7, SIGLEC19P, SIGLECP2, p75, p75 / AIRM1 , sialic acid binding Ig like lectin 7, and AIRM-1 .

[0168] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is Siglec-14. Siglec-14 is also known in the state of the art as sialic acid binding Ig like lectin 14.

[0169] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is Siglec-9. Siglec-9 is also known in the state of the art as SIGLEC9, CD329, CDw329, FOAP-9, OBBP-LIKE, siglec-9, and sialic acid binding Ig like lectin 9.

[0170] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is Siglec-6. Siglec-6 is also known in the state of the art as SIGLEC6, CD327, CD33L, CD33L1 , CD33L2, CDW327, OBBP1 , and sialic acid binding Ig like lectin 6.

[0171] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CD300a. CD300a is also known in the state of the art as CLM-8, CMRF- 35-H9, CMRF-35H, CMRF35-H, CMRF35-H9, CMRF35H, CMRF35H9, IGSF12, IRC1 , IRC1 / IRC2, IRC2, IRp60, CD300a molecule, and CMRF35-like molecule 8.

[0172] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is FcyRIIB. FcyRIIB is also known in the state of the art as FCGR2B, CD32, CD32B, FCG2, FCGR2, IGFR2, Fc fragment of IgG receptor lib, FcRII-c, and FCGR2C. Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is NKG2A. NKG2A is also known in the state of the art as NKG2, NKG2-A, NKG2-B, CD159a, and CD159.

[0173] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CD72. CD72 is also known in the state of the art as Lyb-2, CD72 Molecule, CD72b, B-Cell Differentiation Antigen CD72, and LYB2.

[0174] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CTLA-4. CTLA-4 is also known in the state of the art as CTLA4, ALPS5, CD, CD152, CELIAC3, CTLA-4, GRD4, GSE, IDDM12, and cytotoxic T-lymphocyte associated protein 4.

[0175] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is LAIR1 . LAIR1 is also known in the state of the art as CD305, LAIR-1 , and leukocyte associated immunoglobulin like receptor 1 .

[0176] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is ILT-2. ILT-2 is also known in the state of the art as LILRB1 , CD85J, ILT2, LIR-1 , LIR1 , MIR-7, MIR7, PIR-B, PIRB, and leukocyte immunoglobulin like receptor B1.

[0177] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is ILT-4. ILT-4 is also known in the state of the art as LILRB2, ILT4, MIR10, LIR2, MIR-10, LIR-2, leukocyte immunoglobulin like receptor B2, and CD85D.

[0178] Suitably, the target protein comprises an inhibitory receptor protein, wherein the inhibitory receptor protein is CD31. CD31 is also known in the state of the art as PECAM1 , CD31 / EndoCAM, GPIIA', PECA1 , PECAM-1 , endoCAM, platelet and endothelial cell adhesion molecule 1 , and PCAM-1.

[0179] Suitably, the target protein comprises SIRPa. Signal Regulatory Protein Alpha (also known as SHPS1 , SIRP, BIT, P84, MFR, SHPS-1 , CD172a, PTPNS1 , MYD-1 , Tyrosine-Protein Phosphatase Non-Receptor Type Substrate 1 , CD172 Antigen-Like Family Member A, Inhibitory Receptor SHPS-1 and Macrophage Fusion Receptor) is an immunoglobulin-like cell surface receptor for CD47. SIRPa acts as a docking protein and induces translocation of PTPN6, PTPN11 and other binding partners from the cytosol to the plasma membrane. SIRPa supports adhesion of cerebellar neurons, neurite outgrowth and glial cell attachment. SIRPa is involved in the negative regulation of receptor tyrosine kinase-coupled cellular responses induced by cell adhesion, growth factors or insulin and mediates negative regulation of phagocytosis, mast cell activation and dendritic cell activation. CD47 binding prevents maturation of immature dendritic cells and inhibits cytokine production by mature dendritic cells.

[0180] Suitably, the target protein comprises a member of the carcinoembryonic antigen-related adhesion molecules (CEACAM) family. The CEACAM family are a subgroup of the carcinoembryonic antigen family, found in a range of different cell types and organs. They can act as intercellular adhesions molecules for e.g. bacteria and soluble antigens. CEACAMs are involved in a number of different processes including cell adhesion, proliferation, differentiation and tumour suppression. The CEACAM family includes CEACAM1 (also known as CD66a, Cell-CAM 105, BGP, biliary glycoprotein, and NCA-160); CEACAM 3 (also known as CD66d and CGM1); CEACAM4 (also known as CGM7); CEACAM6 (also known as CD66c, NCA, NCA-90, and CGM6); CEACAM7 (also known as CGM2)_ and CEACAM8 (also known as CD66b, CGM6, CGM8, and NCA-95). Some CEACAMs such as CEACAM1 , CEACAM5 and CEACAM6 are highly associated with cancer and are recognised as valid clinical markers for certain cancer forms.

[0181] Suitably, the target protein comprises a member of the SLAM family of surface receptors. Signaling lymphocytic activation molecule (SLAM) is a family of receptor closely related to the CD2 family of the immunoglobulin (Ig) superfamily of molecules. The SLAM family currently includes nine members named SLAM, CD48, CD229, 2B4, CD84, NTB-A, CRACC, BLAME, and CD2F-10. In general, SLAM molecules possess two to four extracellular Ig domains, a transmembrane segment, and an intracellular tyrosine-rich region. The molecules are differentially expressed on a variety of immune cell types. Several are self ligands and SLAM has been identified as the human measles virus receptor. Several small SH2- containing adaptor proteins are known to associate with the intracellular domains of SLAM family members. For example, in T and NK cells, activated SLAM family receptors become tyrosine phosphorylated and recruit the adaptor known as SAP (SLAM-associated protein) and subsequently the Src kinase Fyn, the ensuing signal transduction cascade influences the outcome of T cell-antigen presenting cell and NK cell-target cell interactions.

[0182] Suitably, the target protein comprises a member of the KIR family of surface receptors, killer cell immunoglobulin-like receptors (KIR; also known as CD158) are a family of transmembrane glycoproteins expressed on NK cells and a subset of T cells.5 The KIR are key regulators of the development, tolerance and activation of NK cells. The KIR family is encoded by 14 highly polymorphic genes (2DL1 to 2DL5, 3DL1 to 3DL3, 2DS1 to 2DS5, and 3DS1), and distinct family members can transduce either activating or inhibitory signals. Nomenclature of KIR is based upon the number of C2-type immunoglobulin-like domains in the extracellular region (2D for two domains, 3D for three domains) and by the length of the cytoplasmic domain (L for long-tailed receptors and S for short ones). Inhibitory KIR have long cytoplasmic domains possessing immunoreceptor tyrosine-based inhibitory motifs (ITIMs), which recruit protein tyrosine phosphatases that are critical for mediating inhibitory function. In contrast, KIR with short cytoplasmic domains associate with a transmembrane signalling adaptor protein, DAP12 (also called KARAP). Consistent with antigen receptor signalling, DAP12-dependent activation occurs through the recruitment of Syk / ZAP-70 tyrosine kinases by immunoreceptor tyrosinebased activation motifs (ITAMs).The only exception to this short / long-tailed rule is KIR2DL4, which is a unique long-tailed activating KIR. Compared with other KIR family members, 2DL4 is only expressed on CD56high NK cells, functions as a more potent activator of cytokine production rather than cytotoxicity, and associates with ITAM-containing FcsRI-y adaptor instead of DAP12.

[0183] Suitably, the target protein comprises a cytokine receptor. As used herein, the term “cytokine receptor" refers to cell-surface glycoproteins that bind specifically to cytokines and transduce their signals. Cytokine receptors may be both membrane-bound and soluble. Cytokine receptors enable cells to communicate with the extracellular environment by responding to cytokines generated in the vicinity or in other parts of the organism. Cytokine receptors may be characterised on the basis of their three-dimensional structure: type I cytokine receptor, type II cytokine receptor, immunoglobulin superfamily cytokine receptors, tumour necrosis factor receptor family, chemokine receptors, and TGF-beta receptor family. Non-limiting examples of type I cytokine receptors include type 1 interleukin receptors, erythropoietin receptor, GM-CSF receptor, G-CSF receptor, growth hormone receptor, prolactin receptor, oncostatin M receptor, and leukaemia inhibitory factor receptor. Non-limiting examples of type II cytokine receptors include type II interleukin receptors, interferon-alpha / beta receptor, and interferon-gamma receptor. Non-limiting examples of immunoglobulin superfamily cytokine receptors include interleukin-1 receptor, CSF1 , C-kit receptor, and interleukin-18 receptor. Non-limiting examples of tumour necrosis factor receptor family include CD27, CD30, CD40, CD120, and lymphotoxin beta receptor. Non-limiting examples of chemokine receptors include lnterleukin-8 receptor, CCR1 , CXCR4, MCAF receptor, and NAP-2 receptor. Non-limiting examples of TGF-beta receptor family include TGF beta receptor 1 and TGF beta receptor 2.

[0184] Suitably, the target protein comprises a growth factor receptor. As used herein, the term "growth factor receptor" refers to a cell surface receptor that binds a growth factor. Growth factors bind to the growth factor receptor to activate the growth factor receptors and initiate a signalling cascade which results in cell growth and / or division. The majority of growth factor receptors comprise receptor tyrosine kinases and the binding of the growth factor to the extracellular ligand binding site leads to the autophosphorylation of tyrosine residues in the intracellular domain and subsequent intracellular signalling. Non-limiting examples of intracellular signalling pathways used by growth factor receptors include the JAK / STAT, MAP kinase, and PI3 kinase pathways. Non-limiting examples of growth factor receptors include the wnt receptor, tie, neurotrophin receptor, ephrin receptor, insulin-like growth factor receptor (IGF receptor), epidermal growth factor receptor (EGF receptor), fibroblast growth factor receptor (FGF receptor), platelet-derived growth factor receptor (PDGF receptor) and vascular endothelial growth factor receptor (VEGF receptor).

[0185] Suitably, the target protein comprises an interferon (IFN) receptor. The IFN family includes two main classes of related cytokines: type I IFNs and type II IFN. There are many type I IFNs, all of which have considerable structural homology. These include IFN-a (which can be further subdivided into 13 different subtypes, IFN-a1 , -a2, -a4, -a5, -a6, -a7, -a8, -a10, -a13, -a14, - a16, -a17 and -a21), IFN-p, IFN-6, IFN-s, IFN-K, IFN-T and IFN-co. II type I IFNs bind a common cell-surface receptor, which is known as the type I IFN receptor. There is only one type II IFN, IFN-y. IFN-y binds to the type II IFN receptor. A relatively new class of IFNs or IFN-like molecules has emerged, the IFN-A molecules: IFN-A1 , -A2 and -A3, which are also known as interleukin-29 (IL-29), IL-28A and IL-28B, respectively. IFN-A molecules bind to IFNLR1 (also known as IL-28 receptor-a, IL-28Ra) and IL-1 ORp.

[0186] The type I IFN receptor is composed of two subunits, IFNAR1 and IFNAR2, which are associated with the Janus activated kinases (JAKs) tyrosine kinase 2 (TYK2) and JAK1 , respectively. Activation of the JAKs that are associated with the type I IFN receptor results in tyrosine phosphorylation of STAT2 (signal transducer and activator of transcription 2) and STAT1 ; this leads to the formation of STAT1-STAT2-IRF9 (IFN-regulatory factor 9) complexes, which are known as ISGF3 (IFN-stimulated gene (ISG) factor 3) complexes. These complexes translocate to the nucleus and bind IFN-stimulated response elements (ISREs) in DNA to initiate gene transcription. For the type I IFN receptor, the IFNAR1 subunit is constitutively associated with tyrosine kinase 2 (TYK2), whereas IFNAR2 is associated with JAK1 .The type II IFN receptor is composed of two subunits, IFNGR1 and IFNGR2, which are associated with JAK1 and JAK2, respectively, the type II IFN receptor, the IFNGR1 subunit associates with JAK1 , whereas IFNGR2 is constitutively associated with JAK2.

[0187] The initial step in both type-l- and type-ll-IFN-mediated signalling is the activation of these receptor-associated JAKs, which occurs in response to a ligand-dependent rearrangement and dimerization of the receptor subunits, followed by autophosphorylation and activation of the associated JAKs. As well as activation of classical JAK-STAT (signal transducer and activator of transcription)-signalling pathways (discussed later), activation of IFN-receptor- associated JAKs seems to regulate, either directly or indirectly, several other downstream cascades. Such diversity of signalling is consistent with the pleiotropic biological effects of IFNs on target cells and tissues.

[0188] Both type I and type II IFNs also induce the formation of STAT1-STAT1 homodimers that translocate to the nucleus and bind GAS (IFN-y-activated site) elements that are present in the promoter of certain ISGs, thereby initiating the transcription of these genes. The consensus GAS element and ISRE sequences are shown.

[0189] The heterodimeric receptor used by type III IFNs to signal is different from the receptor used by type I IFN and consist of the IFNLR1 chain (a.k.a. IL-28Ra) and the IL-10R2 chain. Expression of IFNLR1 is not ubiquitous but appears limited to cells of epithelial origins (e.g., hepatocytes, intestinal cells and lung) and to a specific subset of immune cells (i.e., NK cells, pDCs, and DCs). Engagement of the IFN-A receptor complex by any of the four ligands leads to activation of the receptor-associated tyrosine kinases JAK1 and TYK2, which then phosphorylate specific tyrosines in the intracellular domain of the receptor. This event creates docking sites for STAT1 and STAT2 signalling molecules, which leads to their recruitment and subsequent phosphorylation. The phosphorylated STATs recruit IFN regulatory factor 9 (IRF9), which together form IFN-stimulated gene factor s (ISGF3), which enters the nucleus and drives the transcription of IFN-stimulated genes (ISGs).

[0190] Suitably, the target protein comprises a Type I IFN receptor. Suitably, the target protein comprises a Type II IFN receptor. Suitably, the target protein comprises a Type III IFN receptor.

[0191] Suitably, the target protein comprises a tumour necrosis factor (TNF) receptor. TNF was identified as a product of lymphocytes and macrophages that causes the lysis of certain cell types, particularly tumor cells. TNF and similar molecules were later found to form a TNF superfamily. The receptors for these ligands also form a TNF receptor (TNFR) superfamily. TNFR-like receptors are type 1 transmembrane proteins that use a scaffold of disulfide bridges to form elongated structures. These form “cysteine-rich” domains containing 40 amino acid repeats with three intrachain disulfides made up of six highly conserved cysteines. The functional receptor is a trimer and these elongated structures fit into the grooves between individual chains of the ligand trimer forming a 3:3 complex. The cytoplasmic domains of TNFRs are fairly short and provide docking sites for two major classes of signaling molecules. These are TNFR-associated factors (TRAFs) and “death domain” molecules. Binding of TNF to TNFR1 recruits TNFR1 -associated death domain protein followed by several other signaling molecules (FAS-associated death domain protein, TRAF-2, and the death domain kinase, receptor interacting protein), generally leading to caspase activation and other aspect of apoptosis. Two forms of TNF receptor have been identified, type I receptor (TNFRI), also known as TNF-R55, and type II receptor (TNFRII), also known as TNF-R75, both of which are membrane proteins that bind to TNFa and TNFp and mediate intracellular signaling. Type I, are expressed on all cell types, and type II, are expressed only on cells of the immune system and endothelial cells. At the cellular level, these receptors activate the pathways leading to the activation of transcription factors NF-KB and AP-1 , apoptosis and proliferation, and mitogen activated protein kinases.

[0192] Suitably, the target protein comprises a Type I TNF receptor. Suitably, the target protein comprises a Type II TNF receptor.

[0193] Operably linked and linkers

[0194] Suitably, the first and second regions of the multivalent protein are operably linked. By “operably linked” or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein in the context of the multivalent protein, refers to polypeptides on a single protein molecule that are functionally associated. Thus, a first polypeptide or protein (the first region) that is operably linked to a second polypeptide or protein (the second region) means a situation when the first polypeptide or protein (the first region) is placed in a functional relationship with the second polypeptide or protein (the second region). The first region and second region may be operably linked through a linker interposed between the first and second region.

[0195] Suitably, the multivalent protein comprises a linker interposed between the first and second region. Suitable methods of linking the first region and second region will be familiarto persons skilled in the art, for example using a linker peptide or synthetic compound linker. However, there is no particular limitation on the linkers that can be used in the multivalent proteins described herein.

[0196] The linker peptide may be characterised as flexible or rigid and is based on the amino acid composition of the linker peptide. For example, a flexible linker peptide may comprise amino acids having relatively small side chains, and which may be hydrophilic. Without limitation, the flexible linker peptide may comprise a stretch of glycine (G) and / or serine (S) residues. Examples of such flexible linker peptides include: GS; GGS; SGG; GGGS (SEQ ID NO. 114); SGGG (SEQ ID NO. 134); GGGGS (SEQ ID NO. 140); SGGGG (SEQ ID NO. 135); GGGGGS (SEQ ID NO. 136); SGGGGG (SEQ ID NO. 137); GGGGGGS (SEQ ID NO. 138); SGGGGGG (SEQ ID NO. 139); (GGGGSer)n (SEQ ID NO. 140), (GGGSS)n wherein n is an integer of one or more (SEQ ID NO. 141); and (SGGGG)n, wherein n is an integer of one or more (SEQ ID NO. 142). Suitably, the linker peptide may be modified such that the amino acid sequence GSG (that occurs at the junction of traditional G / S linker polypeptide repeats) is not present. Rigid linker peptides may be utilised to keep a fixed distance between the first and second region to separating the first and second region more efficiently and ensure their independent functions are maintained. Rigid linker peptides may contain, for example, more sterically hindering amino acid side chains, such as (without limitation) tyrosine, histidine and / or proline. In general, rigid linkers exhibit relatively stiff structures by adopting a-helical structures or by containing multiple Pro residues. The a-helical structure is rigid and stable, with intra-segment hydrogen bonds and a closely packed backbone. Therefore, the stiff a-helical linkers can act as rigid spacers between protein domains. Example of such rigid linker peptides include alpha helix-forming linkers with the sequence of (EAAAK)n and a proline-rich sequence, (XP)n, wherein n is an integer of one or more and X designating any amino acid, preferably A, K, or E. Example of such rigid linker peptides include XP; XPXP; XPXPXP; XPXPXPXP; XPXPXPXPXP; XPXPXPXPXPXP; XPXPXPXPXPXPXP; XPXPXPXPXPXPXP; XPXPXPXPXPXPXPXP, wherein X designating any amino acid, preferably A, K, or E.

[0197] Suitably, the linker peptide sequence may include about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. Suitably, the linker peptide sequence may include about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. Suitably, the linker peptide sequence may include about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. Suitably, the linker peptide sequence may include about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. The linker may be covalently linked to the first and second region via, for example, recombinant fusion.

[0198] Suitably, the length and amino acid composition of the linker peptide sequence may be optimised to vary the orientation and / or proximity of a first region and a second region relative to one another to achieve a desired activity of the multivalent protein. Suitably, the orientation and / or proximity of a first region and second region relative to one another may be varied using a “tuning” tool to achieve a tuning effect that would enhance or reduce the agonist activity of the multivalent protein. Suitably, the orientation and / or proximity of a first region and a second region relative to one another may be optimised to create a partial agonist to full agonist versions of the multivalent protein.

[0199] Suitably, the multivalent protein comprises a linker interposed between the first and second regions, wherein the linker comprises or consists of a linker peptide selected from GGGSLEVLFQGPGSGS (SEQ ID NO. 104); AAAYPYDVPDYGSGGGSLEVLFQGPGSGS (SEQ ID NO. 105); AAAYPYDVPDYGSGGGSL (SEQ ID NO. 107); GGGS (SEQ ID NO. 114); GGKLLEVLFQGPGGGS (SEQ ID NO. 115) or a functional variant thereof. Suitably, the multivalent protein comprises a linker interposed between the first and second regions, wherein the linker comprises or consists of linker peptide GGGSLEVLFQGPGSGS (SEQ ID NO. 104) or a functional variant thereof. Suitably, the multivalent protein comprises a linker interposed between the first and second regions, wherein the linker comprises or consists of linker peptide AAAYPYDVPDYGSGGGSLEVLFQGPGSGS (SEQ ID NO. 105) or a functional variant thereof. Suitably, the multivalent protein comprises a linker interposed between the first and second regions, wherein the linker comprises or consists of linker peptide AAAYPYDVPDYGSGGGSL (SEQ ID NO. 107) or a functional variant thereof. Suitably, the multivalent protein comprises a linker interposed between the first and second regions, wherein the linker comprises or consists of linker peptide GGGS (SEQ ID NO. 1 14) or a functional variant thereof. Suitably, the multivalent protein comprises a linker interposed between the first and second regions, wherein the linker comprises or consists of linker peptide GGKLLEVLFQGPGGGS (SEQ ID NO. 115) or a functional variant thereof..

[0200] The linker may be protease-cleavable or non-cleavable. Examples of linkers comprising a 3C protease cleavage site are SEQ ID NO. 104, and 115.

[0201] Suitably, the first and / or second region comprise a protease cleavage site. For example, the multivalent proteins described herein may include at least one protein cleavage site. For example, the multivalent proteins described herein may include a serine protease recognition site. For example, the multivalent proteins described herein may include a cysteine protease recognition site For example, the multivalent proteins described herein may include a 3C, TEV, PreScission, Thrombin, Xa, Enterokinase, or Trypsin protein cleavage site. The inclusion of protein cleavage sites may allow for the multivalent proteins to be turned off. For example, when in use the multivalent proteins may be exposed to cognate protease which cleaves the multivalent protein and reduces or inhibits the activity of the multivalent protein. This may be used to help reduce off target effects or to reduce multivalent protein activity after a desired effect has been achieved.

[0202] Suitably, the linker is a synthetic compound linker such as, for example, a chemical crosslinking agent. Non-limiting examples of suitable cross-linking agents that are commercially available include N-hydroxysuccinimide (NHS), disuccinimidylsuberate (DSS), bis(sulfosuccinimidyl)suberate (BS3), dithiobis(succinimidylpropionate) (DSP), dithiobis(sulfosuccinimidylpropionate) (DTSSP), ethyleneglycol bis(succinimidylsuccinate) (EGS), ethyleneglycol bis(sulfosuccinimidylsuccinate) (sulfo-EGS), disuccinimidyl tartrate (DST), disulfosuccinimidyl tartrate (sulfo-DST), bis[2-

[0203] (succinimidooxycarbonyloxy)ethyl]sulfone (BSOCOES), and bis[2- (sulfosuccinimidooxycarbonyloxy)ethyl]sulfone (sulfo-BSOCOES). Other examples of alterative structures and linkages suitable for the multivalent proteins and multivalent antibodies of the disclosure include those described in Spiess et al., Mol. Immunol. 67:95-106, 2015.

[0204] Binding domain

[0205] Suitably, the at least one of the first and / or second region of the multivalent protein each comprise a binding domain for binding to the effector and / or target proteins.

[0206] Suitably, the first region of the multivalent protein comprises a binding domain for binding to the effector protein and the second region of the multivalent protein comprises a binding domain for binding to the target protein. Accordingly, the first region of the multivalent protein binds to the effector protein and the second region of the multivalent protein binds to the target protein.

[0207] Alternatively, the first region of the multivalent protein comprises a binding domain for binding to the target protein and the second region of the multivalent protein comprises a binding domain for binding to the effector protein. Accordingly, the first region of the multivalent protein binds to the target protein and the second region of the multivalent protein binds to the effector protein.

[0208] Suitably the first region may include a first portion of a binding domain for binding to a target protein and a first portion of a binding domain for binding to a target protein and the second region may include a second portion of the binding domain for binding to a target protein and a second portion of the binding domain for binding to a target protein.

[0209] Suitably, the first region may include a binding domain for an effector protein and a portion of a binding domain for a target protein and the second region includes a second portion of the binding domain for binding the target protein. Suitably, the first region may include a binding domain for an target protein and a portion of a binding domain for a effector protein and the second region includes a second portion of the binding domain for binding the effector protein.

[0210] Suitably, each binding domain comprises an antigen-binding domain or a protein-binding ligand. Suitably, each binding region comprises an antigen-binding domain.

[0211] As used herein, the term “antigen-binding domain” refers to a region or fragment of the binding domain of the multivalent protein that binds to the effector and / or target protein antigen, and which confers antigen specificity to the binding domains. Suitably, the antigen-binding domains bind to an epitope of one or more antigens of the effector and / or target protein. As used herein, the term “epitope” refers to a part of the effector and / or target protein antigens which is specifically recognised by the antigen-binding domains. Epitopes generally consist of chemically active surface groupings of molecules such as amino acids or carbohydrate or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope may be "linear" or "conformational."

[0212] The epitope may be of any chemical nature, including without limitation, peptides, carbohydrates, lipids, glycopeptides and glycolipids. The epitope may be at least substantially the same as a naturally occurring epitope. It may be identical to a naturally occurring epitope, or a modified form of a naturally occurring epitope. Generally, binding molecules (such as the binding domains of a multivalent protein disclosed herein) for a particular protein antigen will preferentially recognise an epitope on the protein antigen in a complex mixture of proteins and / or macromolecules. Suitably, the epitope of the target and / or effector proteins are membrane-proximal or membrane-distal. Suitably, the epitope of the target and / or effector proteins are membrane-proximal. Suitably, the epitope of the target and / or effector proteins are membrane-distal.

[0213] Suitably, a binding domain binds to the effector or target protein with a binding affinity, for example with a dissociation constant (Kd) of 1 x 106M or less, e.g., about 1 x 107M or less, about 1 x 108M or less, about 1 x 109M or less, about 1 x 10wM or less, or about 1 x 1011M or less. The inventors observed that many binding regions of the tested multivalent protein bound the target and / or effector protein with sub nM binding affinities (Table 3) indicating a strong affinity of the binding domains to the target and / or effector proteins.

[0214] Suitably, the antigen-binding domain may be selected from the group consisting of a VHH domain, a single-chain variable fragment (scFv), an antigen-binding fragment (Fab), Fab’, a VH domain, a VL domain, a single domain antibody (dAb), a VNAR domain, or a functional fragment of any and variations thereof. Suitably, the antigen-binding domain may be selected from a VHH domain and a single-chain variable fragment (scFv).

[0215] As used herein, the term “scFv” refers to single chain Fv molecules (scFv), wherein a VH domain and a VL domain are linked by a peptide linker which allows the two domains to associate to form a single antigen binding site (Bird et al., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883. The VH domain and VL domain can be from any class of immunoglobulin (IgG, IgM, IgD, IgE, IgA and IgY) or subclass (e.g. IgG 1 , lgG2, lgG3, lgG4, lgA1 and lgA2) from any species. For example, the species may be, but not limited to dogs, cats, horses, cows, pigs, guinea pigs, mice, rats and the like. The species may be a primate (e.g. a non-human primate). In a preferred example, the species is a human. A scFv can be in either orientation from N- to C-terminus: VH-linker-VL or VL-linker-VH. Typical scFv linkers are well known in the art, are generally 10 to 25 amino acids in length and include glycines and serines. The skilled person would appreciate that there are a number of linkers which could be utilised. Non-limiting examples of these said linkers include peptide linkers comprising or consisting of SEQ ID NO. 106, 110, 1 11 , 112, and 113 or a functional equivalent thereof.

[0216] As used herein, the term “scFv diabody” refers to proteins consisting of two scFvs. As used herein, the term “bispecific T cell engager (BiTE)” refers to proteins consisting of two scFvs of different antibodies which are employed to form a link between T cells and another target.

[0217] Examples of the scFv domains (VH and VL) used in the multivalent protein of the invention include SEQ ID NO. 3, 6, 7, 8, 9, 10, 11 , 12, 13, 17, 25, 27, 28, 29, 30, 31 , 32, 33, 34, and 35. These domains are linked together using one of SEQ ID NO. 106, 110, 111 , 1 12, and 1 13 linkers to form the scFv with an antigen binding site to bind effector and / or target proteins.

[0218] Non-limiting examples of scFv used in the multivalent protein of the invention include SEQ ID NO. 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , and 52.

[0219] Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 3. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 6. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 7. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 8. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 9. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 10. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 11 . Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 12. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 13. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 14. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 17. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 25. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 27. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 28. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 29. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 30. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 31. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 32. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 33. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 34. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 35. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 36. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 37. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 38. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 39. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 40. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 41 . Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 42. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 43. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 44. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 45. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 46. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 47. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 48. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 49. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 50. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 51. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 52.

[0220] The N- to C-terminal orientation of the scFv used in the invention is outlined in Table 2; however, the skilled person would appreciate that switching the orientation of the subdomains and / or using different linkers would also provide scFv capable of being utilised in this invention. For example, scFv (SEQ ID NO. 38) contains scFv subdomains in the orientation of SEQ ID NO. 6 - 110 - 7 and scFv (SEQ ID NO. 39) contains scFv subdomains in the orientation of SEQ ID NO. 7 - 110 - 6.

[0221] As used herein, the terms “VHH” and “VHH domain” are interchangeable and refer to a heavychain antibody devoid of light chains and consisting of only one heavy chain variable region. VHH is one of the smallest antigen-binding domains with complete function. VHH domains are also known as Nanobodies®. VHH can be from any class of immunoglobulin (IgG, IgM, IgD, IgE, IgA and IgY) or subclass (e.g. lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2) from any species. For example, the species may be, but not limited to dogs, cats, horses, cows, pigs, guinea pigs, mice, rats and the like. The species may be a primate (e.g. a non-human primate). In a preferred example, the species is a human.

[0222] Examples of VHH used in the multivalent protein of the invention include SEQ ID NO. 1 , 2, 5, 14, 15, 20, 21 , 22, 23, and 24.

[0223] Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 1 . Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 2. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 5. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 14. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 15. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 20. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 21 . Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 22. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 23. Suitably, the multivalent protein or at least one binding domain thereof may include at least the CDRs as set forth in SEQ ID NO 24.

[0224] Suitably, the antigen-binding domain may comprise an antibody mimetic. As used herein, the term “antibody mimetic” refers to a molecule that is not derived from an antibody or antibody fragment which has the ability to bind to a target antigen. The antibody mimetic could be a polypeptide or fragment thereof of a naturally occurring protein. Alternatively, the antibody mimetic could be a synthetic protein. Antibody mimetics can be classified into two structural classes, namely domain-sized compounds (at 6-20 kDa molecular weight) and constrained peptides (2-4 kDa). Examples of domain-sized scaffolds include Affibodies, Affilins, Anticalins, Atrimers, DARPins, FN3 scaffolds (e.g., Adnectins and Centyrins), Fynomers, Kunitz domains, Pronectins and OBodies. Examples of constrained peptides include Avimers, minibinder, cyclic peptides, bicyclic peptides, and Cys-knots. The skilled person would appreciate that there is a number of antibody mimetics that can be employed so long as the resulting polypeptide includes at least one binding region which specifically binds to the target antigen on a target cell.

[0225] Suitably, the antibody mimetic may be selected from DARPin, Anticalin®, affibody, minibinder, cyclic peptide, or bicyclic peptide.

[0226] Suitably, the antibody mimetic may be a DARPin. DARPins (designed ankyrin repeat proteins) are originated from natural ankyrin proteins and has a structure comprising at least 2 ankyrin repeat motifs, for example, comprising at least 3, 4 or 5 ankyrin repeat motifs of which the most N- and the most C-terminal modules are referred to as "caps", since they shield the hydrophobic core of the protein. The number of internal repeat motifs is indicated as number (e.g. N1 C, N2C, N3C, ...) while the caps are indicated with "N" or "C", respectively. DARPins may have any suitable molecular weight depending on the number of repeat motifs. For example, a DARPin including 3, 4 or 5 ankyrin repeat motifs may have a molecular weight of about 10 kDa, about 14 kDa, or about 18 kDa, respectively. DARPins include a core part that provides structure and a target binding portion that resides outside of the core and binds to a target. The structural core includes a conserved amino acid sequence, and the target binding portion includes an amino acid sequence that differs depending on the target.

[0227] Suitably, the antibody mimetic may be an Anticalin®. Anticalin® proteins are single-chain proteins derived from extracellular human lipocalins which bind with high specificity and potency to antigens. Characteristic for Anticalin® proteins is their barrel structure formed by eight antiparallel p-strands pairwise connected by loops and an attached a-helix. The main structure of Anticalin® proteins is identical to wild type human lipocalins.

[0228] Suitably, the antibody mimetic may be an affibody. Affibody proteins are small (about 6.5 kDa) proteins which can bind to antigens and is based on a three alpha helix bundle domain framework of the natural receptor for the Fc portion of IgG. In some embodiments, an affibody includes a protein scaffold based on the Z domain (the immunoglobulin G binding domain) of protein A, and in contrast to antibodies, affibody proteins are composed of alpha helices and lack disulfide bridges. Methods for engineering and producing affibody proteins are known, and include those described in Nord et al., “A combinatorial library of an a-helical bacterial receptor domain.” Prot. Eng. 1995; 8 (6): 601 -608 and Nord et al., “Binding proteins selected from combinatorial libraries of an a-helical bacterial receptor domain.” Nature Biotechnol. 1997; 15 (8): 772-777. Suitably, the antibody mimetic may be a minibinder. Minibinders are de novo-designed synthetic proteins capable of binding to an antigen. An example of a minibinder is the LCB1 minibinder, which binds the SARS-CoV-2 Spike protein and was de novo-designed using a docking-based approach.

[0229] Suitably, the antibody mimetic may be a cyclic peptide. Cyclic peptides are peptides comprising a cyclic ring structure. The ring structure can be formed through a connection between the amino and carboxyl ends of the peptide (for example, cyclosporin); a connection between the amino end and a side chain (for example, bacitracin); the carboxyl end and a side chain (for example, colistin); or two side chains or more complicated arrangements (for example, alpha-amanitin). The cyclic peptide may be synthetic or derived from a naturally occurring peptide. The length of cyclic peptides ranges from just two amino acid residues to hundreds. Cyclic peptides may be classified into two major categories: homodetic and heterodetic. Homodetic cyclic peptides are those in which the ring is composed exclusively of peptide bonds (i.e. between the alpha carboxyl of one residue to the alpha amine of another). Heterodetic cyclic peptides contain diverse functional groups (at least one non-alpha amide linkage) used to connect the amino acids. These can then be further classified into four types: head-to-tail, head-to-side chain, side chain-to-tail, and side chain-to-side chain cyclisation. Compared to linear peptides, the loop structure endows cyclic peptides with more rigidity, reducing the entropic cost of the receptor-binding. Therefore, cyclic peptides can bind to target molecules with high affinity and specificity.

[0230] Suitably, the antibody mimetic may be a bicyclic peptide. Bicyclic peptides are peptides comprising two cyclic ring structures. Example structures of bicyclic peptides include peptides comprising two loops resulting from a scaffold anchored at three points within the peptide sequence, cyclic peptides containing an internal bridge, double-stapled, and double- macrocyclic peptides. Internal bridges include Phe-Phe, Phe-Tyr, and Tyr-Tyr-like biaryl linkages, tryptophan cross-coupling, bromotryptophan cross-coupling, and cysteine and isoindoles linkages. Each ring in this bicyclic structure can function independently, allowing these peptides to be bifunctional. In contrast to cyclic peptides, bicyclic peptides have stronger conformational restrictions, improving their target binding ability and specificity.

[0231] Suitably, the antigen-binding domain may comprise a T cell receptor (TCR) binding subunit. As used herein, the term “T cell receptor (TCR) binding subunit” refers to Ca, Cp, Va, and / or Vp subunit or fragments thereof that are capable of binding to an antigen in the context of MHC (i.e., the antigen / MHC complex). The antigen / MHC complex may bind to the Va and / or Vp subunits through the CDR1 , CDR2, and / or CDR3 of the Va (CDRa1-CDRa3), and / or Vp (CDRp1-CDRp3) subunits and surrounding framework regions. Typically, the antigen / MHC complex will be on the cell surface of the target protein. The TCR binding subunit may be in the context of a scaffold protein. Suitably, at least one antigen-binding domain may comprise an a TCR Va protein or functional fragment thereof. Suitably, at least one antigen-binding domain may comprise an a TCR Vp protein or functional fragment thereof.

[0232] Suitably, at least one antigen-binding domain may comprise an a TCR like polypeptide. “TCR- like polypeptide” refers to a polypeptide that behaves similarly to a T cell receptor (TCR) in that it specifically binds to an MHC-bound peptide. Suitably, a TCR-like polypeptide is a single chain TCR (see e.g., U.S. Patent Application Publication No. 2012 / 0252742; PCT International Patent Application Publication Nos. WO 1996 / 013593, WO 1999 / 018129, and WO 2004 / 056845; U.S. Pat. No. 7,569,664).

[0233] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 1 and 2, 1 and 14, 1 and 15, 1 and 20, 1 and 21 , 1 and 22, 1 and 23, 1 and 24, 1 and 49, 1 and 50, 1 and 51 , or 1 and 52.

[0234] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 5 and 2, 5 and 14, 5 and 15, 5 and 20, 5 and 21 , 5 and 22, 5 and 23, 5 and 24, 5 and 49, 5 and 50, 5 and 51 , or 5 and 52.

[0235] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 36 and 2, 36 and 14, 36 and 15, 36 and 20, 36 and 21 , 36 and 22, 36 and 23,

[0236] 36 and 24, 36 and 49, 36 and 50, 36 and 51 , or 36 and 52.

[0237] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 37 and 2, 37 and 14, 37 and 15, 37 and 20, 37 and 21 , 37 and 22, 37 and 23,

[0238] 37 and 24, 37 and 49, 37 and 50, 37 and 51 , or 37 and 52.

[0239] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 38 and 2, 38 and 14, 38 and 15, 38 and 20, 38 and 21 , 38 and 22, 38 and 23,

[0240] 38 and 24, 38 and 49, 38 and 50, 38 and 51 , or 38 and 52.

[0241] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 39 and 2, 39 and 14, 39 and 15, 39 and 20, 39 and 21 , 39 and 22, 39 and 23,

[0242] 39 and 24, 39 and 49, 39 and 50, 39 and 51 , or 39 and 52.

[0243] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 40 and 2, 40 and 14, 40 and 15, 40 and 20, 40 and 21 , 40 and 22, 40 and 23,

[0244] 40 and 24, 40 and 49, 40 and 50, 40 and 51 , or 40 and 52. In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 41 and 2, 41 and 14, 41 and 15, 41 and 20, 41 and 21 , 41 and 22, 41 and 23,

[0245] 41 and 24, 41 and 49, 41 and 50, 41 and 51 , or 41 and 52.

[0246] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 42 and 2, 42 and 14, 42 and 15, 42 and 20, 42 and 21 , 42 and 22, 42 and 23,

[0247] 42 and 24, 42 and 49, 42 and 50, 42 and 51 , or 42 and 52.

[0248] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 43 and 2, 43 and 14, 43 and 15, 43 and 20, 43 and 21 , 43 and 22, 43 and 23,

[0249] 43 and 24, 43 and 49, 43 and 50, 43 and 51 , or 43 and 52.

[0250] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 44 and 2, 44 and 14, 44 and 15, 44 and 20, 44 and 21 , 44 and 22, 44 and 23,

[0251] 44 and 24, 44 and 49, 44 and 50, 44 and 51 , or 44 and 52.

[0252] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 45 and 2, 45 and 14, 45 and 15, 45 and 20, 45 and 21 , 45 and 22, 45 and 23,

[0253] 45 and 24, 45 and 49, 45 and 50, 45 and 51 , or 45 and 52.

[0254] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 46 and 2, 46 and 14, 46 and 15, 46 and 20, 46 and 21 , 46 and 22, 46 and 23,

[0255] 46 and 24, 46 and 49, 46 and 50, 46 and 51 , or 46 and 52.

[0256] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 47 and 2, 47 and 14, 47 and 15, 47 and 20, 47 and 21 , 47 and 22, 47 and 23,

[0257] 47 and 24, 47 and 49, 47 and 50, 47 and 51 , or 47 and 52.

[0258] In some embodiments, the multivalent protein comprises amino acid sequences according to SEQ ID NOs: 48 and 2, 48 and 14, 48 and 15, 48 and 20, 48 and 21 , 48 and 22, 48 and 23,

[0259] 48 and 24, 48 and 49, 48 and 50, 48 and 51 , or 48 and 52.

[0260] Suitably, the multivalent proteins described herein include a SpyTag / SpyCatcher system. Suitably, the first region and / or second region or the antigen binding domains thereof each may include one of a SpyTag or SpyCatcher protein.

[0261] The SpyTag / SpyCatcher system is a technology for irreversible conjugation of recombinant proteins. The peptide SpyTag (13 amino acids) spontaneously reacts with the protein SpyCatcher (12.3 kDa) to form an intermolecular isopeptide bond between the pair.

[0262] The sequence of SpyCatcher is provided below: GAMVDTLSGLSSEQGQSGDMTIEEDSATHIKFSKRDEDGKELAGATMELRDSSG KTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHI (SEQ ID NO: 132)

[0263] The sequence of SpyTag is provided below:

[0264] AHIVMVDAYKPTK (SEQ ID NO: 133).

[0265] For example, the first region may include a SpyTag peptide and the second region may include a SpyCatcher peptide. For example, the second region may include a SpyTag peptide and the first region may include a SpyCatcher peptide.

[0266] The inclusion of SpyTag / SpyCatcher peptides may allow the assembly of the first and second regions into a multivalent protein as described herein.

[0267] In some examples, antigen binding domains of the first and / or second regions may include or also include SpyTag / SpyCatcher peptides in order to link together multiple antigen binding domains.

[0268] Antigen-binding domain for binding to the effector protein

[0269] Suitably, the invention provides an antigen-binding domain for binding to the effector protein. In one embodiment, the effector protein is selected from CD2, CD4, CD8, CD14, CD24, CD48, CD55, and / or CD59.

[0270] Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of any one of SEQ ID NO. 2, 14, 15, 20, 21 , 22, 23, 24, 49, 50, 51 , or 52 or a functional fragment thereof.

[0271] SEQ ID NO. 2, 14, 15, 20, 21 , 22, 23, and 24 are VHH domains. In one embodiment, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 as set forth in a VHH domain disclosed herein. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 2. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 14. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 15. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 20. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 21. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 22. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 23. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 24.

[0272] SEQ ID NO. 49, 50, 51 , and 52 are scFvs. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in an scFv domain disclosed herein.

[0273] Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 49. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 50. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 51 . Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 52.

[0274] Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 49. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 50. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 51 . Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 52.

[0275] Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 49. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 50. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 51 . Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 52.

[0276] Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 49. Suitably, the antigenbinding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 50. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 51. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 52.

[0277] Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 2 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 14 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 15 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 20 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 21 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 22 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 23 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 24 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 49 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 50 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 51 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of SEQ ID NO. 52 or a functional fragment thereof.

[0278] Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 2, 14, 15, 19, 20, 21 , 22, 23, 24, 49, 50, 51 , 52 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 2 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 14 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 15 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 23 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 24 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 51 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 52 or a functional fragment thereof.

[0279] The term “sequence identity” as used herein refers to the degree of sequence matching between two nucleic acid sequences or two amino acid sequences as determined using the algorithm of for example Karlin & Attschul (1990) Proc. Natl. Acad. Sci. 87: 2264-2268, modified as in Karlin & Attschul (1993) Proc. Natl. Acad. Sci. 90: 5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Attschul et al. (1990) T. Mol. Biol. Q15: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, wordlength=12, to obtain nucleotide sequences homologous to a nucleic acid molecule of the invention. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a reference amino acid sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Attschul et al. (1997) Nucl. Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g. XBLAST and NBLAST) are used. Other algorithms, programs and default settings may also be suitable such as, but not only, the GCG-Sequence Analysis Package of the U.K. Human Genome Mapping Project Resource Centre that includes programs for nucleotide or amino acid sequence comparisons.

[0280] Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD4, comprises or consists of any one of SEQ ID NO. 22, 23, or 24 or a functional fragment thereof.

[0281] SEQ ID NO. 22, 23, and 24 are VHH domains.

[0282] Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD4, comprises or consists of SEQ ID NO. 22 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD4, comprises or consists of SEQ ID NO. 23 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD4, comprises or consists of SEQ ID NO. 24 or a functional fragment thereof.

[0283] Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD4, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 22, 23, or 24 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD4, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 22 or a functional fragment thereof. Suitably, the antigenbinding domain for binding to the effector protein, wherein the effector protein is CD4, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 23 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD4, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 24 or a functional fragment thereof.

[0284] Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of any one of SEQ ID NO. 2, 14, 15, 20, 21 , 49, or 50 or a functional fragment thereof. SEQ ID NO. 2, 14, 15, 20, and 21 are VHH domains.

[0285] SEQ ID NO. 49 and 50 are scFvs.

[0286] Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 2 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 14 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 15 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 20 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 21 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 49 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD8, comprises or consists of SEQ ID NO. 50 or a functional fragment thereof.

[0287] Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 2, 14, 15, 20, 21 , 49, or 50 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 2 or a functional fragment thereof. Suitably, the antigenbinding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 14 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 15 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 20 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 21 or a functional fragment thereof. Suitably, the antigenbinding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 49 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD8, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 50 or a functional fragment thereof.

[0288] Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD48, comprises or consists of any one of SEQ ID NO. 51 or 52 or a functional fragment thereof.

[0289] SEQ ID NO. 51 and 52 are scFvs.

[0290] Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD48, comprises or consists of SEQ ID NO. 51 or a functional fragment thereof. Suitably, the antigen-binding domain for specifically binding to the effector protein, wherein the effector protein is CD48, comprises or consists of SEQ ID NO. 52 or a functional fragment thereof.

[0291] Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD48, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 51 or 52 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD48, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 51 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the effector protein, wherein the effector protein is CD48, comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 52 or a functional fragment thereof. Antigen-binding domain for binding to the target protein

[0292] Suitably, the invention provides an antigen-binding domain for binding to the target protein. In one embodiment, the target protein is PD-1 , BTLA, CD200R1 , CD5, VISTA, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, CD66, LAIR1 , ILT-2, ILT-4, and / or CD31 .

[0293] Suitably, the antigen-binding domain for binding to the target protein comprises or consists of any one of SEQ ID NO. 1 , 5, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48 or a functional fragment thereof.

[0294] SEQ ID NO. 1 , and 5are VHH domains.

[0295] Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 1. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, and HCDR3 sequences of SEQ ID NO. 5.

[0296] SEQ ID NO. 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, and 48 are scFvs.

[0297] Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 37. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 38. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 39. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 40. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 41 . Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 42. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 43. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 44. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 45. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 46. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 47. Suitably, the antigen-binding domain comprises the HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 48.

[0298] Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 36. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 37. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 38. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 39. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 40. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 41 . Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 42. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 43. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 44. Suitably, the antigenbinding domain comprises a variable heavy chain as set forth in SEQ ID NO 45. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 46. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 47. Suitably, the antigen-binding domain comprises a variable heavy chain as set forth in SEQ ID NO 48.

[0299] Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 36. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 37. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 38. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 39. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 40. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 41. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 42. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 43. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 44. Suitably, the antigenbinding domain comprises a variable light chain as set forth in SEQ ID NO 45. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 46. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 47. Suitably, the antigen-binding domain comprises a variable light chain as set forth in SEQ ID NO 48.

[0300] Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 36. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 37. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 38. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 39. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 40. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 41 . Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 42. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 43. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 44. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 45. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 46. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 47. Suitably, the antigen-binding domain comprises a VHH comprising the HCDR1 , HCDR2, HCDR3, or LCDR1 , LCDR2, and LCDR3 as set forth in SEQ ID NO 48.

[0301] Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 1 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 5 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 36 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 37 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 38 or a functional fragment thereof. Suitably, the antigenbinding domain for binding to the target protein comprises or consists of SEQ ID NO. 39 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 40 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 41 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 42 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 43 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 44 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 45 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 46 or a functional fragment thereof. Suitably, the antigen-binding domain for binding to the target protein comprises or consists of SEQ ID NO. 47 or a functional fragment thereof. Suitably, the antigenbinding domain for binding to the target protein comprises or consists of SEQ ID NO. 48 or a functional fragment thereof.

[0302] Suitably, the antigen-binding domain for binding to the target protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 1 , 5, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 45, 46, 47, 48 or a functional fragment thereof.

[0303] Suitably, the antigen-binding domain for binding to the target protein, wherein the target protein is PD-1 , comprises or is derived from any one of SEQ ID NO. 1 , 5, 36, 37, 38, 39, 40, 41 , 42, 43, 48 or a functional fragment thereof.

[0304] Suitably, the antigen-binding domain for binding to the target protein, wherein the target protein is PD-1 comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 1 , 5, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 45, 46, 47, 48 or a functional fragment thereof.

[0305] Suitably, the antigen-binding domain for binding to the target protein, wherein the target protein is BTLA, comprises or is derived from any one of SEQ ID NO. 45, 46, 47 or a functional fragment thereof.

[0306] Suitably, the antigen-binding domain for binding to the target protein, wherein the target protein is BTLA, comprises or consists of an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 45, 46, 47 or a functional fragment thereof.

[0307] Suitably, the antigen-binding domain for binding to the target protein, wherein the target protein is CD200R1 , comprises or is derived from SEQ ID NO. 44 or a functional fragment thereof.

[0308] Suitably, the antigen-binding domain for binding to the target protein, wherein the target protein is CD200R1 comprises or consists of an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 44 or a functional fragment thereof. Protein-binding ligand

[0309] As used herein, the term “protein-binding ligand” refers to a molecule which binds to a target protein molecule to form a complex. Protein-binding ligand binding occurs via intermolecular forces, such as ionic bonds, hydrogen bonds, and Van der Waals forces. The interaction between the protein-binding ligand and target protein is typically reversible, but occasionally may be irreversible. Protein-binding ligands include substrates, inhibitors, activators, and neurotransmitters. Protein-binding ligands are essential for a variety of biological processes, such as interactions in antibody-antigen recognition, cell-cell communication, and signal transduction. An exemplary protein-binding ligand is PD-L1 , which binds to receptor PD-1 to form the complex PD-1 / PD-L1. Other example protein-binding ligands include cytokines, growth factors, receptor extracellular domain (ECD) of cell surface receptors, or a functional variant of any thereof. Suitably, the protein-binding ligand comprises a cytokine, a growth factor, a receptor extracellular domain (ECD) of cell surface receptor, or a functional variant of any thereof.

[0310] Suitably, the protein-binding ligand comprises a cytokine or a functional variant thereof. As used herein, the term “cytokine” is a generic term referring to small (typically <30 kDa) proteins, peptides, and glycoproteins released by one cell population that act on another cell through a cell surface receptor to modulate immune responses. Cytokines are secreted in response to infection, inflammation, trauma, sepsis, cancer, and reproduction to stimulate, recruit, and proliferate immune cells. Cytokines include interleukins (IL), chemokines, interferons, and tumour necrosis factors (TNF). Suitably, the protein-binding ligand comprises a cytokine selected from one or more of IL, chemokine, interferon, and / or TNF. Examples of cytokines are IL-1 (IL-1a, IL-1 p, IL-1 Ra, and IL-1 R2), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL- 9, IL-11 , IL-12, IL 13, IL-15 ... IL-35, IFN-y, and TNF-a. Suitably, the protein-binding ligand comprises a cytokine selected from one or more of IL-1 (IL-1 a, IL-1 p, IL-1 Ra, and IL-1 R2), IL- 2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-11 , IL-12, IL 13, IL-15 ... IL-35, IFN-y, and / or TNF-a. Autoimmune diseases may be caused in part by cytokine- and chemokine-mediated dysregulation of Th cell subset differentiation.

[0311] Suitably, the protein-binding ligand comprises a growth factor or a functional variant thereof. As used herein, the term “growth factor” a generic term referring to secreted proteins and hormones acting on a target cell through a cell surface receptor to cause one or more of the following responses in the target cell: growth including proliferation, differentiation, survival, regeneration, migration, regain of function, improvement of function. Growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic proteins (BMPs), ciliary neurotrophic factors, colony-stimulating factors (CSF), epidermal growth factor (EGF), ephrins, erythropoietin (EPO), fibroblast growth factors (FGF), foetal bovine somatotrophin (FBS), GDNF family of ligands (GFL), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin, Insulin-like growth factors, keratinocyte growth factor (KGF), migration-stimulating factor (MSF), macrophage-stimulating protein (MSP) (also known as hepatocyte growth factor-like protein (HGFLP)), myostatin (GDF-8), neuregulins, neurotrophins, placental growth factor (PGF), platelet-derived growth factor (PDGF), renalase (RNLS), T-cell growth factor (TCGF), thrombopoietin (TPO), transforming growth factors, and vascular endothelial growth factor (VEGF). The FGF family includes FGF1 , FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11 , FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21 , FGF22, and FGF23. Ciliary neurotrophic factors include ciliary neurotrophic factor (CNTF) and leukaemia inhibitory factor (LIF). Colony-stimulating factors include macrophage colony-stimulating factor (M-CSF), granulocyte colony-stimulating factor (G- CSF), and granulocyte macrophage colony-stimulating factor (GM-CSF). GDNF family of ligands include glial cell line-derived neurotrophic factor (GDNF), neurturin, persephin, and artemin. Insulin-like growth factors include insulin-like growth factor-1 (IGF-1) and insulin-like growth factor-2 (IGF-2). Neuregulins include neuregulin 1 (NRG1), neuregulin 2 (NRG2), neuregulin 3 (NRG3), and neuregulin 4 (NRG4). Neurotrophins include brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin- 4 (NT-4). Transforming growth factors include transforming growth factor alpha (TGF-a) and transforming growth factor beta (TGF-p).

[0312] Suitably, the protein-binding ligand comprises a receptor extracellular domain (ECD) of cell surface receptor or a functional variant thereof. As used herein, the term “receptor extracellular domain (ECD) of cell surface receptor” refers to an extracellular domain of a cell surface receptor protein which is capable of acting as a ligand for a receptor.

[0313] Suitably, the protein-binding ligand comprises a peptidic hormone, “peptidic hormone” refers to a class of peptides or proteins that have endocrine functions in living animals. Typically, peptidic hormones exert their functions by binding to receptors on the surface of target cells and transmitting signals via intracellular second messengers. Exemplary peptidic hormones include Adiponectin i.e. Acrp30; Adrenocorticotropic hormone (or corticotropin) i.e. ACTH; Amylin (or Islet Amyloid Polypeptide) i.e. IAPP; Angiotensinogen and angiotensin i.e. AGT; Anti-Mullerian hormone (or Mullerian inhibiting factor or hormone) i.e. AMH; Antidiuretic hormone (or vasopressin, arginine vasopressin) i.e. ADH; Atrial-natriuretic peptide (or atriopeptin) i.e. ANP; Brain natriuretic peptide i.e. BNP; Calcitonin i.e. CT; Cholecystokinin i.e. CCK; Corticotropin-releasing hormone i.e. CRH; Cortistatin i.e. CORT; Endothelin i.e.; Enkephalin i.e.; Erythropoietin i.e. EPO; Follicle-stimulating hormone i.e. FSH; Galanin i.e. GAL; Gastric inhibitory polypeptide i.e. GIP; Gastrin i.e. GAS; Ghrelin i.e.; Glucagon i.e. GCG; Glucagon-like peptide-1 i.e. GLP1 ; Gonadotropin-releasing hormone i.e. GnRH; Growth hormone i.e. GH or hGH; Growth hormone-releasing hormone i.e. GHRH; Guanylin i.e. GN; Hepcidin i.e. HAMP; Human chorionic gonadotropin i.e. hCG; Human placental lactogen i.e. HPL; Inhibin i.e.; Insulin i.e. INS; Insulin-like growth factor (or somatomedin) i.e. IGF; Leptin i.e. LEP; Lipotropin i.e. LPH; Luteinizing hormone i.e. LH; Melanocyte stimulating hormone i.e. MSH or a-MSH; Motilin i.e. MLN; Orexin i.e.; Osteocalcin i.e. OCN; Oxytocin i.e. OXT; Pancreatic polypeptide i.e. Parathyroid hormone i.e. PTH; Pituitary adenylate cyclase- activating peptide i.e. PACAP; Prolactin i.e. PRL; Prolactin releasing hormone i.e. PRH; Relaxin i.e. RLN; Renin i.e.; Secretin i.e. SCT; Somatostatin i.e. SRIF; Thrombopoietin i.e. TPO; Thyroid-stimulating hormone (or thyrotropin) i.e. TSH; Thyrotropin-releasing hormone i.e. TRH; Uroguanylin i.e. UGN; or Vasoactive intestinal peptide i.e. VIP, or an isoform, homolog, fragment, variant or derivative of any of these proteins.

[0314] Suitably, the protein-binding ligand comprises a GPCR ligand. “GPCR ligand” refers to a hydrophobic or lipophilic moiety that has biological activity (either agonist or antagonist) at a G protein-coupled receptor (GPCR). GPCR ligands are physically and chemically diverse and can include: photons; ions (H+, Zn++, Ca++, etc.); odorants; tastants; vitamins (e.g. niacin, vitamin A1 aldehyde, etc.); peptidic and non-peptidergic hormones (estrogen, angiotensin, etc.); proteins (e.g. chemokines), neurotransmitters (dopamine, serotonin, etc.); natural products (morphine, salvinorin A, etc.); intermediary metabolites (ATP, ADP, fatty acids, bile acids, etc.); and products from human commensal bacteria.

[0315] In some examples, the first and / or second region or the antigen binding domains thereof include one or more GPCR ligands. For example, in addition to antigen binding domains for binding to one or more targets as described herein and one or more effectors as described herein, the multivalent proteins or antigen binding domains thereof may additionally include one or more GPCR ligands.

[0316] In some examples, the effector may be GPCR.

[0317] Multivalent protein constructs

[0318] Suitably, the multivalent protein construct may comprise or consist of a sequence according to any one of SEQ ID NO. 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 122, 123, 124, or 125 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 53 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 54 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 55 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 56 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 57 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 58 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 59 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 60 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 61 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 62 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 63 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 64 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 65 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 66 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 67 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 68 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 69 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 70 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 71 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 72 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 73 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 74 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 75 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 76 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 122 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 123 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 124 or a functional fragment thereof. Suitably, the multivalent protein construct may comprise or consist of a sequence according to SEQ ID NO. 125 or a functional fragment thereof.

[0319] Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 122, 123, 124, or 125 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 53 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 54 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 55 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 56 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 57 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 58 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 59 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 60 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 61 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 62 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 63 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 64 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 65 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 66 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 67 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 68 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 69 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 70 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 71 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 72 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 73 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 74 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 75 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 76 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 122 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 123 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 124 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 125 or a functional fragment thereof.

[0320] Suitably, the multivalent protein may comprise an N- and / or C-terminal purification-tag. A purification-tags is a peptide sequence suitable for the purification or identification of a multivalent protein. Typically, a purification tag specifically binds to another moiety with affinity for the purification tag. Such moieties which specifically bind to a purification tag are usually attached to a matrix or a resin, such as agarose beads. Moieties which specifically bind to purification tags include antibodies, other proteins (e.g. Protein A or Streptavidin), nickel or cobalt ions or resins, biotin, amylose, maltose, and cyclodextrin. Moieties which specifically bind to purification tags include antibodies, other proteins (e.g. Protein A or Streptavidin), nickel or cobalt ions or resins, biotin, amylose, maltose, and cyclodextrin. Exemplary purification-tags include the myc tag (EQKLISEEDL, SEQ ID NO. 143), the Strep tag (WSHPQFEK, SEQ ID NO. 144), the Flag tag (DYKDDDDK, SEQ ID NO. 145), the V5 tag (GKPIPNPLLGLDST, SEQ ID NO. 146), the HA tag (YPYDVPDYA, SEQ ID NO. 147) and polyhistidine-tags (which will bind to metal ions such as nickel or cobalt ions) and functional variants thereof. Suitably, the multivalent protein may comprise an N- and / or C-terminal polyhistidine-tag. Suitably, the multivalent protein may comprise an N-terminal polyhistidine- tag. Suitably, the multivalent protein may comprise a C-terminal polyhistidine-tag. Typically, the polyhistidine-tag comprises several histidine residues. Suitably, the polyhistidine-tag comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11 , or at least 12 histidine residues. Suitably, the polyhistidine-tag comprises 8 histidine residues. Such polyhistidine-tag is known as an “octa histidine-tag”, “8xHis-tag”, or “His8 tag”. Suitably, the multivalent protein may comprise or consist of a C- terminal polyhistidine-tag comprising or consisting of SEQ ID NO. 118, 119, 120 ora functional variant thereof. When the moiety is an antibody, the purification tags are known as epitope tags and can be used to facilitate detection and / or purification of the multivalent polyprotein. Suitably, the multivalent protein may comprise or consist of an N- and / or C-terminal epitopetag. Suitably, the multivalent protein may comprise or consist of an N-terminal epitope-tag. Suitably, the multivalent protein may comprise or consist of a C-terminal epitope-tag. Suitably, the multivalent protein may comprise or consist of a C-terminal epitope-tag comprising or consisting of SEQ ID NO. 117 or 121 or a functional variant thereof.

[0321] Suitably, the multivalent protein may comprise or consist of a sequence according to any one of SEQ ID NO. 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 126, or 127 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 77 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 78 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 79 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 80 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 81 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 82 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 83 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 84 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 85 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 86 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 87 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 88 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 89 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 90 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 91 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 92 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 93 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 94 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 95 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 96 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 97 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 98 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 99 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 100 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 101 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 102 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 103 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 126 or a functional fragment thereof. Suitably, the multivalent protein may comprise or consist of a sequence according to SEQ ID NO. 127 or a functional fragment thereof.

[0322] Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to any one of SEQ ID NO. 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 101 , 102, 103, 126, or 127 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 77 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 78 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 79 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 80 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 81 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 82 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 83 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 84 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 85 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 86 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 87 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 88 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 89 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 90 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 91 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 92 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 93 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 94 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 95 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 96 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 97 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 98 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 99 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 100 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 101 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO.

[0323] 102 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 103 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 126 or a functional fragment thereof. Suitably, the multivalent protein comprises or consists of an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO. 127 or a functional fragment thereof. Multivalent Antibodies

[0324] The multivalent proteins described herein may be include a single polypeptide chain or two or more polypeptide chains associated with each other.

[0325] For example, a single polypeptide chain may encode both the first and second regions. The second region may be linked to the first region by a protein or amino acid linker that is continuous with the amino acids encoding the first and second regions, thus forming a single chain protein encoding both the first and second regions described herein.

[0326] Suitably, the multivalent proteins may be formed from two distinct polypeptide chains that are linked by a chemical linker that links the two polypeptide chains in a continuous fashion (i.e. in line with the two polypeptide chains). Such linked polypeptide chains may be also be referred to single chain polypeptides.

[0327] In other examples, multivalent proteins described herein may include two or more polypeptide chains linked by inter-chain bonding or interactions. Therefore, forming a multivalent protein having a first and second polypeptide chain. Each of the first and second polypeptide chains may encode a portion of the first region, a portion of one or more effector antigen binding domains, a portion of the second region and / or a portion of one or more target antigen binding domains. For example, the first polypeptide chain may encode a first portion of the effector antigen binding domain and a first portion of the target antigen binding domain; and the second polypeptide chain encoding a second portion of the effector antigen binding domain and a second portion of the target antigen binding domain. The two chains may then be joined to each other, for example by inter-chain bonding, such as via disulphide bonding (e.g. via cysteine residues).

[0328] Suitably, each of the first and / or second regions include an Fc region. Suitably, each of the first and second regions include an Fc region. Suitably, each of the first and / or second polypeptide chains include an Fc region. Suitably, each of the first and second polypeptide chains include an Fc region. The Fc region may be any suitable Fc region known in the art.

[0329] Fc region refers to a C-terminal region of an immunoglobulin heavy chain, including nativesequence Fc regions and variant Fc regions. Although the boundaries of the Fc region of an immunoglobulin heavy chain might vary, the human IgG heavy-chain Fc region is usually defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl-terminus thereof. The C-terminal lysine (residue 447 according to the EU numbering system) of the Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Suitable native-sequence Fc regions that may be used for the multivalent proteins described herein include human lgG1 , lgG2 (lgG2A, lgG2B), lgG3 and lgG4. A “variant Fc region” or “engineered Fc region” comprises an amino acid sequence that differs from that of a native-sequence Fc region by virtue of at least one amino acid modification, preferably one or more amino acid substitution(s). Suitably, the variant Fc region has at least one amino acid substitution compared to a native-sequence Fc region or to the Fc region of a parent polypeptide, e.g., from about one to about ten amino acid substitutions. Suitably, the variant Fc region will possess at least about 80% homology with a native-sequence Fc region. Suitably, the Fc region has been modified by the choice of expression host, enzymatic treatment of amino acid substitutions to have reduced glycosylation and binding to FcyR, relative to the native protein. Examples of variant Fc regions and modifications that may be included can be found in Hale, G., De Vos, J., Davy, A. D., Sandra, K., & Wilkinson, I. (2024). Systematic analysis of Fc mutations designed to reduce binding to Fc-gamma receptors. mAbs, 16(1), 2402701 . https: / / doi.org / 10.1080 / 19420862.2024.2402701 van der Horst, Hilma J et al. “Fc-Engineered Antibodies with Enhanced Fc-Effector Function for the Treatment of B-Cell Malignancies.” Cancers vol. 12,10 3041. 19 Oct. 2020, doi:10.3390 / cancers12103041 and Abdeldaim, Dalia T, and Katharina Schindowski. “Fc- Engineered Therapeutic Antibodies: Recent Advances and Future Directions.” Pharmaceutics vol. 15,10 2402. 28 Sep. 2023, doi:10.3390 / pharmaceutics15102402. Suitably, the Fc region is an inert Fc region. As used herein, the term “inert Fc region” refers to an Fc region with reduced, minimised, or eliminated antibody effector functions such as ADCC (antibody-dependent cell-mediated cytotoxicity), CDC (complement-dependent cytotoxicity), and / or ADCP (antibody-dependent cellular phagocytosis). Inert Fc regions may also be referred to as “effectorless Fc regions”, “Fc-tamed antibodies”, or “Fc-silent”. Inert Fc regions with varying degrees of reduced receptor binding have been developed with the introduction of mutations in the Fc of key amino acids that mediate Fc interactions with complement and Fc receptors. A common design feature of all these formats is that undesirable Fc interactions with Fey receptors and the complement receptor C1 q can be decoupled from binding to the Fc Receptor (FcRn) which conveys the sought after benefit of long serum persistence. As with effector binding, in vivo serum persistence conferred by FcRn is also a tuneable property that can be modulated by mutations in the Fc region. For example, Fc Silent™ antibodies comprise inert Fc regions which comprise point mutations which abrogate binding of the Fc region to Fc receptors (FcyR, FcR). Therefore, their antibody directed cytotoxicity (ADCC) effector function is abolished, but Fc FcRn binding is retained. The multivalent proteins described herein, with or without Fc regions may be referred to a multivalent antibodies. Therefore, in one aspect there is provided a multivalent antibody that comprises a first region and second region as described herein.

[0330] For example, the multivalent proteins described herein may be bi-specific antibodies configured for binding to an effector and target as described herein in cis (i.e. on the same cell). There are more than 100 distinct multivalent antibody formats known in the art. Generally multivalent antibodies fall into two categories: IgG-like (including Fc region or portion thereof) and fragment-based.

[0331] Suitably, the multivalent proteins described herein are single domain multivalent antibodies also referred to as multivalent nanobodies (such as a bispecific nanobody). As used herein, the term "nanobody" has the meaning commonly understood by those skilled in the art and refers to an antibody fragment consisting of a single monomer variable antibody domain (e.g., a single heavy chain variable region), typically derived from a variable region of a heavy chain antibody (e.g., a camelid antibody or a shark antibody). Typically, nanobodies consist of 4 framework regions and 3 complementarity determining regions, having the structure FR1- CDR1 -FR2-CDR2-FR3-CDR3-FR4. Nanobodies may be truncated at the N-or C-terminus such that they comprise only a portion of FR1 and / or FR4, or lack one or both of those framework regions, so long as they substantially retain antigen binding and specificity. “Bispecific nanobody" refers to a bispecific antibody formed from two nanobodies

[0332] Suitably, the multivalent proteins described herein are diabodies. “Diabody refers to a fusion protein or a bivalent antibody which can bind different antigens. A diabody is composed of two single protein chains which comprise fragments of an antibody, namely variable fragments. Diabodies comprise a heavy chain variable domain (VH) connected to a light-chain variable domain (VL) on the same polypeptide chain (VH-VL, or VL-VH). By using a short peptide connecting the two variable domains, the domains are forced to pair with the complementary domain of another chain and thus, create two antigen-binding sites. Diabodies can target the different antigens (bispecific).

[0333] Suitably, the multivalent proteins described herein are Dual-Affinity Re-Targeting molecules (DARTs). DART is a type of bispecific antibody construct developed by MacroGenics. DART is a protein scaffold wherein the VH of a first antigen-binding domain is linked to a VL of a second antigen-binding domain on a second polypeptide, and the VH of the second antigenbinding domain is linked to the VL on the first polypeptide in an arrangement VL(A)- VH(B)+VL(B)-VH(A), wherein an interchain disulfide bond is introduced to stabilize the molecule. In some examples, the DART may include Fc regions (e.g. a DART-Fc). Suitably, the multivalent proteins described herein are CrossMabs. CrossMab variants are based on the inter-arm cross-exchange of bispecific IgG antibodies and heterodimerization of heavy chains via knob-into-hole configuration, thereby ensuring correct chain binding. Fragments may be exchanged at the level of Fab domains (CrossMab-Fab), variable VH-VL domains, and invariant CH1-CL domains. Suitably, the multivalent proteins described herein are CrossMab-Fab.

[0334] Suitably, the multivalent proteins described herein are tandem diabodies. A tandem diabody is an antigen-binding molecule constructed by linking at least four variable domains (two heavy chain variable domains (VH) and two light chain variable domains (VL)) in a single gene construct enabling homo-dimerization of two of the translated polypeptide chains. In such tandem diabodies the linker length is such that it prevents intramolecular pairing of the variable domains so that the molecule cannot fold back upon itself to form a monomeric single-chain molecule, but rather is forced to pair with the complementary domains of another chain. The variable domains are also arranged such that the corresponding variable domains pair during this dimerization (Weichel et al., European Pharmaceutical Review 2015; 20(1):27-32; Reusch et al., mAbs 2014; 6:3, 728-739)..

[0335] Suitably, the multivalent proteins described herein are Tandem ScFvs. Tandem ScFvs consist of two scFvs connected in a single polypeptide chain.

[0336] Suitably, the multivalent proteins described herein are BiTE-Fc (scFv diabody with Fc region).

[0337] In some examples, multivalent proteins described herein may include two or more first regions and second regions that are linked to each other by an Fc region. For example, a multivalent proteins described herein having a first and second region as described herein may be combined with a second multivalent protein as described herein having another (e.g. second) first region and another (e.g. second) second region. Therefore, in one aspect there is provide a multivalent antibody comprising at least two multivalent proteins as described herein. For example, multivalent antibody comprising at least two multivalent proteins as described herein may be a DART molecule, CrossMab-Fab, CrossMabCH1-CL, CrossMab-VH-VL, Tandem CrossFab, DART-FC, BiTE-Fc or duobody as described herein.

[0338] Other multivalent formats that can be used include, for example, KA-bodies, SMIPs, D Ls, strand-exchange engineered domain bodies (SEEDbodies), Affibodies, Fynomers, Kunitz Domains, Albu-dabs, DVD-IG, Covx-bodies, peptibodies, scFv-lgs, SVD-lgs, dAb-lgs, Knobs- in-Holes, peptidic Bicycle, and triomAbs. Exemplary bispecific formats are discussed in Garber et al., Nature Reviews Drug Discovery 73:799-801 (2014), which is herein incorporated by reference in its entirety. Suitably, the multivalent proteins described herein may be a TCR-like antibody.

[0339] “TCR-like antibody,” refers to an antibody or parts thereof that is specific for an epitope-MHC complex. For example, see Denkberg et. al., Direct visualization of distinct T cell epitopes derived from a melanoma tumor-associated antigen by using human recombinant antibodies with MHC restricted T cell receptor-like specificity, PNAS 99 (14) 9421-9426 (2002).

[0340] Expression Constructs and Vectors

[0341] In some examples, there is provided expression constructs that comprise the isolated nucleic acid molecules described herein. "Expression construct" as used herein means a nucleic acid sequence capable of directing expression of a particular nucleic acid sequence in an appropriate host cell, comprising a promoter operably linked to the polynucleotide of interest which is operably linked to termination signal sequences. It also typically comprises sequences required for proper translation of the polynucleotide sequence. The expression construct comprising the polynucleotide of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components. The expression construct may also be one that is naturally occurring but has been obtained in a recombinant form useful for heterologous expression. Suitably, the expression construct is heterologous with respect to the host, i.e., the particular polynucleotide of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event. The expression of the polynucleotide sequence in the expression construct may be underthe control of, for example, a constitutive promoter or of an inducible promoter that initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, such as a plant, the promoter can also be specific to a particular tissue, organ, or stage of development. Expression constructs may be provided with nucleic acid vectors as described below (i.e. expression vectors).

[0342] By “operably linked” or “operably associated” as used herein, it is meant that the indicated elements are functionally related to each other, and are also generally physically related. Thus, the term “operably linked” or “operably associated” as used herein in the context of polynucleotides, refers to polynucleotides on a single nucleic acid molecule that are functionally associated. Thus, a first polynucleotide sequence or nucleic acid molecule that is operably linked to a second polynucleotide sequence or nucleic acid molecule means a situation when the first polynucleotide sequence or nucleic acid molecule is placed in a functional relationship with the second polynucleotide sequence or nucleic acid molecule. Alternatively, a first polypeptide or protein that is operably linked to a polypeptide or protein means a situation when the first polypeptide or protein is placed in a functional relationship with the second polypeptide or protein.

[0343] For instance, a promoter is operably associated with a polynucleotide sequence or nucleic acid molecule if the promoter effects the transcription or expression of said polynucleotide sequence or nucleic acid molecule. Those skilled in the art will appreciate that the control sequences (e.g., promoter) need not be contiguous with the polynucleotide sequence or nucleic acid molecule to which it is operably associated, as long as the control sequences function to direct the expression thereof. Thus, for example, intervening untranslated, yet transcribed, sequences can be present between a promoter and a polynucleotide sequence or nucleic acid molecule, and the promoter can still be considered “operably linked” to or “operatively associated” with the polynucleotide sequence or nucleic acid molecule.

[0344] Also provided herein are nucleic acid vectors or nucleic acid vector systems, which include a nucleic acid molecule as described herein. The nucleic acid vector or nucleic acid vector system may have one or more nucleic acid vectors. The polyproteins that are encoded by the nucleic acid molecules described herein may be encoded by one or more nucleic acid sequences, for example, in a nucleic acid composition. In examples where all of the polyproteins are encoded by a single nucleic acid molecule, the nucleic acid molecule may be present within a single nucleic acid vector (and thus, the nucleic acid vector system described herein may comprise one vector). In examples where the polyproteins are encoded by two or more nucleic acid molecules (wherein the plurality of nucleic acid molecules, together, encode all of the components of a VLP) these two or more nucleic acid molecules may be present within one nucleic acid vector (e.g. in different open reading frames of the vector), or may be distributed over two or more nucleic acid vectors. In this example, the nucleic acid vector system will comprise a plurality of distinct nucleic acid vectors (i.e. nucleic acid vectors with different nucleotide sequences).

[0345] Any appropriate nucleic acid vector can be used. By way of example only, the nucleic acid vector may be a plasmid, a cosmid, or a viral vector, such as a retroviral vector or a lentiviral vector. Adenovirus, adeno-associated virus, vaccinia virus, canary poxvirus, herpes virus, minicircle vectors, and naked (synthetic) DNA / RNA may also be used (for details on minicircle vectors; see, for example, non-viral Sleeping Beauty transposition from, minicircle vectors as published by R Monjezi et al., Leukemia 2017). Alternatively, single-stranded or doublestranded DNA or RNA can be used to transfect host cells (see Roth et al. 2018 Nature vol 559; page 405). As used herein, the term “nucleic acid vector” refers to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been operably linked. The nucleic acid vector can be capable of autonomous replication or integration into a host cell’s DNA. The nucleic acid vector may include restriction enzyme sites for insertion of recombinant DNA and may include one or more selectable markers or suicide genes. The nucleic acid vector can be a nucleic acid molecule in the form of a plasmid, a bacteriophage or a cosmid. Preferably the nucleic acid vector is suitable for expression in a cell (i.e. the nucleic acid vector is an “expression vector”). Preferably, expression in a human cell.

[0346] The nucleic acid vector may comprise regulatory sequences. "Regulatory sequences" as used herein, refers to, DNA or RNA elements that are capable of controlling gene expression. Examples of expression control sequences include promoters, enhancers, silencers, TATA- boxes, internal ribosomal entry sites (IRES), attachment sites for transcription factors, transcriptional terminators, polyadenylation sites etc. Optionally, the nucleic acid vector includes one or more regulatory sequences operatively linked to the nucleic acid sequence to be expressed. Regulatory sequences include those which direct constitutive expression, as well as tissue-specific regulatory and / or inducible sequences.

[0347] In some examples, the nucleic acid vector comprises the nucleic acid sequence of interest operably linked to a promoter. "Promoter", as used herein, refers to the nucleotide sequences in DNA to which RNA polymerase binds to start transcription. The promoter may be inducible or constitutively expressed. Alternatively, the promoter is under the control of a repressor or stimulatory protein. The promoter may be one that is not naturally found in the host cell (e.g. it may be an exogenous promoter). The skilled person in the art is well aware of appropriate promoters for use in the expression of target proteins, wherein the selected promoter will depend on the host cell.

[0348] The nucleic acid vector may comprise a transcriptional terminator. “T ranscriptional terminator” as used herein, refers to a DNA element, which terminates the function of RNA polymerases responsible for transcribing DNA into RNA. Preferred transcriptional terminators are characterized by a run of T residues preceded by a GC rich dyad symmetrical region.

[0349] The nucleic acid vector may comprise a translational control element. “Translational control element”, as used herein, refers to DNA or RNA elements that control the translation of mRNA. Preferred translational control elements are ribosome binding sites. Preferably, the translational control element is from a homologous system as the promoter, for example a promoter and its associated ribozyme binding site. Preferred ribosome binding sites are known, and will depend on the chosen host cell. The nucleic acid vector may comprise a selectable marker. "Selectable marker" as used herein, refers to proteins that, when expressed in a host cell, confer a phenotype onto the cell which allows a selection of the cell expressing said selectable marker gene. Generally this may be a protein that confers a new beneficial property onto the host cell (e.g. antibiotic resistance) or a protein that is expressed on the cell surface and thus accessible for antibody binding. Appropriate selectable markers are well known in the art.

[0350] Preferably the nucleic acid vector comprises those genetic elements which are necessary for expression of polyproteins described herein by a host cell. The elements required for transcription and translation in the host cell include a promoter, a coding region for the protein(s) of interest, and a transcriptional terminator.

[0351] A person of skill in the art will be well aware of the molecular techniques available for the preparation of (expression) vectors and how the (expression) vectors may be transduced or transfected into an appropriate host cell (thereby generating a modified cell described further below). The (expression) nucleic acid vector system described herein can be introduced into cells by conventional techniques such as transformation, transfection or transduction. “Transformation”, “transfection”, and “transduction” generally refer to techniques for introducing a foreign (exogenous) nucleic acid molecules into a host cell and therefore encompass methods such as electroporation, microinjection, gene gun delivery, transfection with retroviral, lentiviral or adeno-associated vectors, lipofection, superfection etc. The specific method used typically depends on both the type of nucleic acid vector and the cell. Appropriate methods for introducing nucleic acid molecules and vectors into host cells such as human cells are well known in the art; see for example Sambrook et al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y; Ausubel et al (1987) Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY; Cohen et al (1972) Proc. Natl. Acad. Sci. USA 69, 2110; Luchansky et al (1988) Mol. Microbiol. 2, 637- 646. Further conventional methods that are suitable for preparing expression vectors and introducing them into appropriate host cells are described in detail in WO2016 / 071758 for example.

[0352] In some examples, the host cell is contacted with the nucleic acid vector system (e.g. viral vector) in vitro, ex vivo, and in some examples, the host cell is contacted with the nucleic acid vector system (e.g. viral vector) in vivo.

[0353] The term "host cell" includes any cell into which the nucleic acid molecule or nucleic acid vector described herein may be introduced. Once a nucleic acid molecule or nucleic acid vector system has been introduced into the cell, it may be referred to as a “modified cell” herein. Once the nucleic acid molecule or nucleic acid vector is introduced into the host cell, the resultant modified cell should be capable of expressing the encoded polyproteins (and e.g. correctly localising the polyproteins protein and processing the polyprotein to form a VLP).

[0354] In some examples, the nucleic acid molecules or nucleic acid vectors may be introduced using CRISPR technology by engineering with CRISPR / Cas9 and homologous directed repair (HDR) or non-homologous end joining (NHEJ) is therefore encompassed. Other conventional methods such as transfection, transduction or transformation of cells may also be used.

[0355] The term “modified cell” refers to a genetically altered (e.g. recombinant) cell. The modified cell includes at least one exogenous nucleic acid molecule (i.e. a nucleic acid sequence that is not naturally found in the host cell). In the context of the invention, the exogenous molecule comprises at least one of the polyprotein component parts described herein or at least one nucleic acid molecule encoding the polyprotein or parts thereof.

[0356] The isolated nucleic acid molecules, expression constructs and nucleic acid vector as described may be included in a delivery vector specific to the administration and delivery of nucleic acid molecules, expression constructs and nucleic acid vector as described to a subject. For example, the nucleic acid molecules, expression constructs and nucleic acid vectors as described, may be encapsulated in lipids, polymers, or dendrimers. For example, nucleic acid molecules, expression constructs and nucleic acid vector as described, may be delivered to a target tissue or host cell by the use of lipid nanoparticles, autologous T cells, CAR-T cells, plasmid DNA, modified CD34+ hHSPCs, cytotoxic T lymphocytes, or T cells. For example, see Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the Messenger: Advances in Technologies for Therapeutic mRNA Delivery. Mol Ther. 2019;27(4):710-728. doi:10.1016 / j.ymthe.2019.02.012 and Qin S, Tang X, Chen Y, et al. mRNA-based therapeutics: powerful and versatile tools to combat diseases. Signal Transduct Target Ther. 2022;7(1):166. Published 2022 May 21. doi:10.1038 / s41392-022-01007-w.

[0357] Examples of delivery vectors that may be used to introduce the nucleic acid molecules, expression constructs and nucleic acid vectors will be known those skilled in the art. Examples include those disclosed in WO2017004143A1 , WO2023196931 A1 , WO2024037578A1 , and WO2023056917A1

[0358] In some examples, the delivery vector comprises a lipid nanoparticle. In some examples, the delivery vector comprises lipofectamineTM. Lipofectamine consists of a 3:1 mixture of DOSPA (2,3-dioleoyloxy-N- [2(sperminecarboxamido)ethyl]-N,N-dimethyl-1 -propaniminium trifluoroacetate) and DOPE, which complexes with negatively charged nucleic acid molecules to allow them to overcome the electrostatic repulsion of the cell membrane. In some examples, the multivalent proteins or parts thereof may be encoded by multiple nucleic acids. For example, a first region may be encoded by a first nucleic acid and the second region may be encoded by a second nucleic acid. In such examples, the nucleic acids may be referred to as a nucleic acid composition. Suitably, the multivalent proteins or parts thereof may be comprises with multiple expression constructs or vectors.

[0359] The host cells, or recombinant cell including at least one nucleic acid molecule encoding a multivalent protein (for example an expression vector) as described herein may be used to produce a multivalent protein as described herein. Suitably, there is provided a method of producing a multivalent protein as described, the method including introducing at least one nucleic acid molecule encoding a multivalent protein (for example an expression vector) as described herein into a recombinant cell and expressing the multivalent protein. The cell may be cultured under conditions suitable for expression of the multivalent protein. For example, the recombinant cell may be induced to express the multivalent protein when the nucleic acid molecule encodes an inducible promoter.

[0360] After expression of the multivalent protein, the multivalent protein may be recovered from the cell. For example, the multivalent protein may be isolated from the cell to provide an isolated multivalent protein. An isolated protein refers to a protein that is substantially free of other proteins, cellular material, separation medium, and culture medium when isolated from cells or produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. If the protein is not secreted into the culture medium, the cells can disrupted and the protein may be isolated from the lysate by known methods. The multivalent proteins described herein may be purified. For example, purified by any method known in the art for purification of a protein, for example, by chromatography (e.g., ion exchange, anionic exchange, affinity, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.

[0361] Medical uses

[0362] Given the above, there is provided herein a method of treating a disease or condition in a subject, the method comprising administering an effective amount of a multivalent protein of the invention, a multivalent antibody of the invention, a nucleic acid of the invention, or a recombinant cell of the invention, to a subject in need thereof.

[0363] There is also provided herein a method for modulating cell signalling mediated by a target protein, wherein the target protein signals through a phosphorylation mechanism in a subject, the method comprising administering an effective amount of a multivalent protein of the invention, a multivalent antibody of the invention, a nucleic acid of the invention, or a recombinant cell of the invention, to a subject in need thereof.

[0364] There is also provided herein a multivalent protein of the invention, a multivalent antibody of the invention, a nucleic acid of the invention, or a recombinant cell of the invention, for use as a medicament.

[0365] There is also provided herein a multivalent protein of the invention, a multivalent antibody of the invention, a nucleic acid of the invention, or a recombinant cell of the invention, for use in treating: a. an autoimmune disease; b. an autoinflammatory disease; c. inflammation; d. transplantation; e. allergies; and / or f. cancer

[0366] Suitably, the multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein may be administered in combination with one or more additional therapeutics. For example, the additional therapeutic may be administered, prior to, subsequently to and / or concurrently with the multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins or composition thereof as described herein.

[0367] The multivalent proteins described herein may help to guide immune response in a subset of cells (such as immune cells) and therefore act as an adjuvant in treatment of diseases that involve or are associated with an immune response. For example, the multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein may be used or for use as an adjuvant in treating infection, autoimmunity, and / or cancer. Suitably, the multivalent protein, the nucleic acid composition, the recombinant cell, the multivalent antibody or the pharmaceutical composition as described herein, are for use an adjuvant in the prophylaxis or treatment of infection, autoimmunity, and / or cancer.

[0368] Infectious Disease

[0369] The multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein may be for use in preventing and / or treating an infectious disease. The medical uses and methods of preventing and / or treating an infectious disease may include administering to a subject in need thereof a therapeutically effective amount of a the multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein.

[0370] “Infectious disease” refers to a disease which results from an infection. An infection is a condition caused by the invasion of an organism by a foreign agent (i.e., infectious agent). Infectious agents include, but are not limited to, bacteria, fungi, viruses, viroids, nematodes (e.g., parasites such as roundworms and pinworms), anthropods (e.g., mites, fleas, lice, ticks), and macroparasites (e.g., tapeworms). Common infectious diseases include bacterial and viral infections.

[0371] The multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein may be particularly useful in prevention or treatment of infectious diseases caused by intracellular pathogens. For example, viruses (e.g., CMV HIV), bacteria (e.g., Listeria, Mycobacteria, Salmonella (e.g., S. typhi) enteropathogenic Escherichia coll (EPEC), enterohaemorrhagic Escherichia coll (EH EC), Yersinia, Shigella, Chlamydia, Chlamydophila, Staphylococcus, Legionella), protozoa (e.g., Taxoplasma), fungi, and intracellular parasites (e.g., Plasmodium (e.g., P. vivax, P. falciparum, P. ovale, and P. malariae).

[0372] In some examples, the methods of preventing and / or treating infectious disease is a method of vaccination.

[0373] "Vaccination" refers to the administration of multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein intended to generate an immune response, for example to a disease-causing pathogen. For Vaccination can be administered before, during, and / or after exposure to a disease-causing pathogen, and in some examples, before, during, and / or shortly after exposure to the agent. In some examples, vaccination includes multiple administrations, appropriately spaced in time, of a composition or formulation as described herein.

[0374] "Treatment” in relation to infectious diseases refers to any administration of a multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or composition thereof as described herein that partially or completely alleviates, ameliorates, relieves, inhibits, delays onset of, reduces severity of and / or reduces incidence of one or more symptoms or features of an infectious disease or the predisposition toward the disease. Such treatment may be of a subject who does not exhibit signs of the relevant disease, and / or of a subject who exhibits only early signs of the disease. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease. As such the term "treating" in reference to infectious diseases refers to the vaccination of a subject. Prevention" refers to a delay of onset of an infectious disease. Prevention may be considered complete when onset of an infectious disease, disorder has been delayed for a predefined period of time.

[0375] Autoimmune disease

[0376] As used herein, the term “autoimmune disease” refers to a disease or disorder (or manifestation thereof or resulting condition therefrom or co-segregate thereof) arising from and directed against a subject’s own tissues resulting from an immune response against a self-antigen on or of a self-tissue or tissue component, mediated by the adaptive immune response (self-antibody responses and cell-mediated responses). Autoimmune disease may be organ-specific and / or systemic in nature. Non-limiting examples of self-antigens (also known as autoantigens in the art) include Rh blood group antigens, platelet integrin GPIIb / llla, non-collagenous domain of basement membrane, collagen type IV, epidermal cadherin, cardiac muscle antigens (due to cross-reactivity with Streptococcal cell-wall antigens), rheumatoid factor IgG complexes (with or without hepatitis C antigens), DNA, histones, ribosomes, snRNP, scRNP, rheumatoid factor IgG complexes, pancreatic beta-cell antigen, synovial joint antigen, myelin basic protein, proteolipid protein, and myelin oligodendrocyte glycoprotein. The skilled person would appreciate that any protein, peptide, carbohydrate, lipid, nucleic acids or combination thereof of a subject may act as a self-antigen. The selfantibody responses are mediated by autoantibodies against self-antigens. Autoantibodies may bind self-antigens on self-cells / tissues / organs resulting in damage, inflammation and / or organ dysfunction. Further, autoantibodies can bind to receptors and stimulate or block its stimulation by its natural ligand. The cell-mediated responses are mediated by auto-reactive T cells against self-antigens. For example, activated CD8+ auto-reactive T cells can kill selfcells via the Fas / Fas ligand (FasL) pathway, or by releasing cytolytic granules at the effector / target cell junction. Activated cytotoxic auto-reactive T lymphocytes (CTLs) can also cause self-tissue damage by secreting high levels of pro-inflammatory cytokines, such as TNFa and IFNy.

[0377] Examples of autoimmune diseases or disorders include arthritis (rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis, and ankylosing spondylitis), psoriasis, dermatitis including atopic dermatitis; chronic idiopathic urticaria, including chronic autoimmune urticaria, polymyositis / dermatomyositis, toxic epidermal necrolysis, systemic scleroderma and sclerosis, responses associated with inflammatory bowel disease (IBD) (Crohn's disease, ulcerative colitis), and IBD with co-segregate of pyoderma gangrenosum, erythema nodosum, primary sclerosing cholangitis, and / or episcleritis), respiratory distress syndrome, including adult respiratory distress syndrome (ARDS), meningitis, IgE-mediated diseases such as anaphylaxis and allergic rhinitis, encephalitis such as Rasmussen's encephalitis, uveitis, colitis such as microscopic colitis and collagenous colitis, glomerulonephritis (GN) such as membranous GN, idiopathic membranous GN, membranous proliferative GN (MPGN), including Type I and Type II, and rapidly progressive GN, allergic conditions, eczema, asthma, conditions involving infiltration of T cells and chronic inflammatory responses, atherosclerosis, autoimmune myocarditis, leukocyte adhesion deficiency, systemic lupus erythematosus (SLE) such as cutaneous SLE, lupus (including nephritis, cerebritis, pediatric, non-renal, discoid, alopecia), juvenile onset diabetes, multiple sclerosis (MS) such as spino-optical MS, allergic encephalomyelitis, immune responses associated with acute and delayed hypersensitivity mediated by cytokines and T-lymphocytes, tuberculosis, sarcoidosis, granulomatosis including Wegener's granulomatosis, agranulocytosis, vasculitis (including Large Vessel vasculitis (including Polymyalgia Rheumatica and Giant Cell (Takayasu's) Arteritis), Medium Vessel vasculitis (including Kawasaki's Disease and Polyarteritis Nodosa), CNS vasculitis, and ANCA-associated vasculitis, such as Churg-Strauss vasculitis or syndrome (CSS), aplastic anemia, Coombs positive anemia, Diamond Blackfan anemia, immune hemolytic anemia including autoimmune hemolytic anemia (AIHA), pernicious anemia, pure red cell aplasia (PRCA), Factor VIII deficiency, hemophilia A, autoimmune neutropenia, pancytopenia, leukopenia, diseases involving leukocyte diapedesis, CNS inflammatory disorders, multiple organ injury syndrome, myasthenia gravis, antigen-antibody complex mediated diseases, anti-glomerular basement membrane disease, anti-phospholipid antibody syndrome, allergic neuritis, Bechet disease, Castleman's syndrome, Goodpasture's Syndrome, Lambert-Eaton Myasthenic Syndrome, Reynaud's syndrome, Sjorgen's syndrome, Stevens-Johnson syndrome, solid organ transplant rejection (including pretreatment for high panel reactive antibody titers, IgA deposit in tissues, and rejection arising from renal transplantation, liver transplantation, intestinal transplantation, cardiac transplantation, etc.), graft versus host disease (GVHD), pemphigoid bullous, pemphigus (including vulgaris, foliaceus, and pemphigus mucus-membrane pemphigoid), autoimmune polyendocrinopathies, Reiter's disease, stiff-man syndrome, immune complex nephritis, IgM polyneuropathies or IgM mediated neuropathy, idiopathic thrombocytopenic purpura (ITP), thrombotic throbocytopenic purpura (TTP), thrombocytopenia (as developed by myocardial infarction patients, for example), including autoimmune thrombocytopenia, autoimmune disease of the testis and ovary including autoimmune orchitis and oophoritis, primary hypothyroidism; autoimmune endocrine diseases including autoimmune thyroiditis, chronic thyroiditis (Hashimoto's Thyroiditis), subacute thyroiditis, idiopathic hypothyroidism, Addison's disease, Grave's disease, autoimmune polyglandular syndromes (or polyglandular endocrinopathy syndromes), Type I diabetes also referred to as insulin-dependent diabetes mellitus (IDDM), including pediatric IDDM, and Sheehan's syndrome; autoimmune hepatitis, Lymphoid interstitial pneumonitis (HIV), bronchiolitis obliterans (non-transplant) vs NSIP, Guillain-Barre Syndrome, Berger's Disease (IgA nephropathy), primary biliary cirrhosis, celiac sprue (gluten enteropathy), refractory sprue with co-segregate dermatitis herpetiformis, cryoglobulinemia, amylotrophic lateral sclerosis (ALS; Lou Gehrig's disease), coronary artery disease, autoimmune inner ear disease (AIED), autoimmune hearing loss, opsoclonus myoclonus syndrome (QMS), polychondritis such as refractory polychondritis, pulmonary alveolar proteinosis, amyloidosis, giant cell hepatitis, scleritis, monoclonal gammopathy of uncertain / unknown significance (MGUS), peripheral neuropathy, paraneoplastic syndrome, channelopathies such as epilepsy, migraine, arrhythmia, muscular disorders, deafness, blindness, periodic paralysis, and channelopathies of the CNS; autism, inflammatory myopathy, and focal segmental glomerulosclerosis (FSGS).

[0378] For example, autoimmune diseases or disorders include, but are not limited to, graft-versus- host disease (GVHD), solid organ transplantation rejection, vasculitis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1 DM), multiple sclerosis (MS), giant cell arteritis (GCA), psoriasis (PsO), psoriatic arthritis (PsA), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), ankylosing spondylitis (AS), Sjogren's syndrome (SjS), autoimmune hepatitis, scleroderma, celiac disease, Addison's disease, Hashimoto's disease, Graves' disease, atrophic gastritis / pernicious anemia, acquired hypogonadism / infertility, hypoparathyroidism, Coombs positive-hemolytic anemia, chronic allergic diseases (such as asthma, hay fever, or allergic rhinitis), Crohn's disease, male or female infertility, Behcet's, Wegener's granulomatosis, myocarditis, myositis, polymyalgia rheumatic (PMR), spontaneous abortion, vitiligo, atherosclerosis, autoimmune pancreatitis, bullous pemphigoid, chronic viral infections, and myasthenia gravis. For purposes of the present disclosure, preferred autoimmune diseases are graft-versus-host disease (GVHD), solid organ transplantation rejection, vasculitis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1 DM), multiple sclerosis (MS), giant cell arteritis (GCA), psoriasis (PsO), psoriatic arthritis (PsA), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), ankylosing spondylitis (AS), Sjogren's syndrome (SjS), autoimmune hepatitis, and scleroderma. For example, autoimmune diseases are graft-versus-host disease (GVHD), solid organ transplantation rejection, vasculitis, systemic lupus erythematosus (SLE), type 1 diabetes mellitus (T1 DM), multiple sclerosis (MS), giant cell arteritis (GCA), psoriasis (PsO), psoriatic arthritis (PsA), rheumatoid arthritis (RA), inflammatory bowel disease (IBD), ulcerative colitis (UC), ankylosing spondylitis (AS), Sjogren's syndrome (SjS), autoimmune hepatitis, and scleroderma.

[0379] The medical uses and methods of preventing and / or treating autoimmune disease may include administering to a subject in need thereof a therapeutically effective amount of a multivalent protein, a multivalent antibody, nucleic acid encoding said multivalent protein, recombinant cell or compositions thereof as described herein.

[0380] Autoinflammatory disease

[0381] As used herein, the term “autoinflammatory disease” refers to a disease or disorder (or manifestation thereof or resulting condition therefrom) arising from and directed against a subject’s own tissues resulting from an immune response against a self-tissue or tissue component mediated by a dysregulated innate immune response. Examples of autoinflammatory diseases or disorders include Hereditary fever syndromes (Familial Mediterranean fever (FMF), Tumour necrosis factor receptor-associated periodic fever syndrome (TRAPS), Hyper-lgD syndrome (H IDS)) , monogenic autoinflammatory syndromes (Cryopyrin-associated periodic syndromes (CAPS), Familial cold autoinflammatory syndrome (FCAS), Muckle-Wells syndrome (MWS), Neonatal onset multisystem inflammatory disease / chronic Infantile neurologic cutaneous arthropathy syndrome (NOMID / CINCA), Syndrome of pyogenic arthritis, pyoderma gangrenosum and acne (PAPA syndrome, PAPAS, PAPGA syndrome), Juvenile systemic granulomatosis (Blau syndrome, early onset sarcoidosis), Deficiency of interleukin-1 receptor antagonist (DIRA), Mevalonic aciduria, Majeed syndrome), Nonhereditary or polygenic disorders (Schnitzler syndrome, Crohn disease, Behcet disease, Hidradenitis suppurativa, Psoriatic arthritis, Syndrome of periodic fever, aphthous stomatitis, pharyngitis and adenitis (Pyoderma gangrenosum and Acne Syndrome (PAPAS), PFAPA syndrome), Systemic-onset juvenile idiopathic arthritis, Adultonset Still disease), Adult Still's Disease (AOSD), Haploinsufficiency of A20 (HA20), Paediatric Granulomatous Arthritis (PGA), PLCG2-Associated Antibody Deficiency and Immunoregulation Dysregulation (PLAID), PLCG2-associated autoinflammation, antibody deficiency, Autoinflammation, antibody deficiency, and immune dysregulation (APLAID), sideroblastic anaemia with B-cell immunodeficiency; periodic fever; and developmental delay (SIFD).

[0382] The medical uses and methods of preventing and / or treating autoinflammatory disease may include administering to a subject in need thereof a therapeutically effective amount of a multivalent protein, a multivalent antibody, nucleic acid encoding said multivalent protein, recombinant cell or compositions thereof as described herein. Inflammation

[0383] As used herein, the term “inflammation” refers to a subject’s response to stimuli, such as infections (bacteria, viruses, parasites), damage to cells or tissue, or irritants. Inflammation is a complex process mediated by a variety of pro-inflammatory or anti-inflammatory stimulators or mediators, which are synthesised and released by cells and tissues. Inflammation can occur in any part of the subject’s body, including the central nervous system (such as encephalitis, myelitis, or meningitis), the peripheral nervous system (such as neuritis), the eye (such as dacryoadenitis, scleritis, episcleritis, or keratitis), the ear (such as otitis), the heart (such as endocarditis, myocarditis, or pericarditis), the vascular system (such as arteritis, phlebitis, or capillaritis), the respiratory system (such as sinusitis, rhinitis, pharyngitis, epiglottitis, laryngitis, tracheitis, bronchitis, pneumonitis, or pleurisy), the digestive system (such as stomatitis, gingivitis, glossitis, tonsillitis, sialadenitis, parotitis, cheilitis, pulpitis, gnathitis, oesophagitis, gastritis, gastroenteritis, enteritis, colitis, pancolitis, appendicitis, cryptitis, hepatitis, cholecystitis, or pancreatitis), the integumentary system (such as dermatitis or mastitis), the musculoskeletal system (such as arthritis, myositis, synovitis, tenosynovitis, or bursitis), the urinary system (such as nephritis, ureteritis, cystitis, or urethritis), the female reproductive system (such as oophoritis, salpingitis, endometritis, myometritis, parametritis, cervicitis, vaginitis, or vulvitis), the male reproductive system (such as orchitis, epididymitis, prostatitis, vasculitis, balanitis, or posthitis), the endocrine system (such as insulitis, hypophysitis, thyroiditis, parathyroiditis, or adrenalitis), or the lymphatic system (such a lymphangitis or lymphadenitis).

[0384] Inflammation may be transient inflammation or chronic inflammation. Transient inflammation lasts from 1 to 14 days. Chronic inflammation lasts for more than 14 days. Once transient inflammation has lasted for more than 14 days, it is considered chronic inflammation which may last several months to years.

[0385] Suitably, the multivalent protein of the invention, multivalent antibodies of the invention, the nucleic acid of the invention or the recombinant cell of the invention or compositions thereof modulates inflammation in the subject. Suitably, the multivalent protein of the invention modulates inflammation in the subject. Suitably, the nucleic acid of the invention modulates inflammation in the subject. Suitably, the recombinant cell of the invention modulates inflammation in the subject. Suitably, the multivalent antibodies of the invention modulates inflammation in the subject. As used herein, the term “modulates inflammation” refers to the act of altering the inflammatory response. This modulation may involve stimulating, suppressing, or otherwise adjusting the activity, duration, or intensity of the inflammatory process. Stimulating the inflammatory process may be achieved through enhancing or amplifying inflammatory responses by stimulating the production or activity of pro- inflammatory mediators such as cytokines (for example, TNF-alpha, IL-6, IL-1 beta). Alternatively, suppressing the inflammatory process may be achieved through inhibiting the production or activity of pro-inflammatory mediators, promoting the release of antiinflammatory mediators (for example, IL-10, TGF-beta), or increasing the function of regulatory immune cells.

[0386] Suitably, the multivalent proteins, multivalent antibodies, nucleic acid encoding said multivalent proteins or compositions as described herein by administered in combination with one or more anti-inflammatory compounds or drugs. Anti-inflammatories include any of a number of compounds, agents, therapeutic mediums or drugs known to those skilled in the art, either steroidal or non-steroidal, and generally characterized has having the property of counteracting or suppressing the inflammatory process. Non-steroidal inflammatory drugs or compounds comprise a class of drugs that shares the property of being analgesic, antipyretic and anti-inflammatory by way of interfering with the synthesis of prostaglandins. Such nonsteroidal anti-inflammatories include, but are not limited to, indomethacin, ibuprofen, naxopren, piroxicam and nabumetone. Anti-inflammatory steroids, include triamcinolone acetonide (generic name) and corticosteroids that include, for example, triamcinolone, dexamethasone, fluocinolone, cortisone, prednisolone, fiumetholone, and derivatives thereof (See also U.S. Pat. No. 5,770,589).

[0387] The medical uses and methods of preventing and / or treating inflammation may include administering to a subject in need thereof a therapeutically effective amount of a multivalent protein, a multivalent antibody, nucleic acid encoding said multivalent protein, recombinant cell or compositions thereof as described herein.

[0388] Allergies

[0389] As used herein, the term “allergy” refers to an immune response to otherwise inert allergens. An allergen is any foreign compound, substance, or material that is capable of evoking an immune response. Allergens may be, among otherthings, natural or native allergens, modified natural allergens, synthetic allergens, recombinant allergens, allergoids, and mixtures or combinations thereof. Examples of naturally occurring allergens include pollen allergens (e.g., tree, weed, herb and grass pollen allergens), mite allergens (from e.g. house dust mites and storage mites), insect allergens (e.g., inhalant, saliva- and venom origin allergens), animal allergens from e.g. saliva, hair and dander from animals (e.g. dog, cat, horse, rat, mouse, etc.), fungi allergens and food allergens. “Allergy symptom” or “allergic reaction” refers to the body's response to an allergen. An allergic reaction can be localized to one area (skin that came into contact with allergen) or generalized. Allergic reactions may include, but are not limited to, rash, itching, hives, swelling, difficulty breathing, wheezing, angioedema, difficulty swallowing, nasal congestion, runny nose, shortness of breath, nausea, stomach cramps, abdominal pain, vomiting and / or low blood pressure.

[0390] Subjects in need of treatment may identified or diagnosed as having an allergic disease by any suitable method known in the art. For example, diagnosis made through any one or more of clinical history, detection of specific IgE molecules in the blood stream (i.e., via blood tests), skin prick testing, patch test methods, challenge test methods or elimination methods (such as elimination diets).

[0391] The medical uses and methods of preventing and / or treating an allergic disease may include administering to a subject in need thereof a therapeutically effective amount of a multivalent protein, a multivalent antibody, nucleic acid encoding said multivalent protein, recombinant cell or compositions thereof as described herein.

[0392] The terms “prevent,” “preventing,” or the like, as used with reference to an allergic reaction or allergic condition, refer to preventing development of allergy, an allergic reaction or an allergic condition. The term, as used herein, also includes reducing or abrogating allergen sensitization to prevent an allergic reaction.

[0393] “Response” of a subject to treatment indicates that the subject manifests a reduction in the clinical symptoms. Clinical symptoms may be assessed over the course of treatment, i.e. symptoms before treatment may be compared to symptoms during and after treatment.

[0394] Alternatively, a reduction in symptoms may be determined by comparison to a baseline level established before treatment. Concerning allergy, this approach is particularly useful where, for example, immunotherapy is carried out in subjects not currently experiencing symptoms, as may be the case for seasonal grass pollen allergy sufferers, who may be treated before the pollen season. Symptoms may be assessed by standard methods, such as patient selfassessment or recording of the amount of medication required. The degree of a subject’s response to treatment may be assessed by measuring the degree of reduction of severity in symptoms.

[0395] Cancers

[0396] The multivalent proteins, multivalent antibodies, nucleic acids encoding said multivalent proteins, recombinant cells or compositions as described herein may be for use in methods of treating cancer. Cancer” refers a broad group of diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division may result in the formation of malignant tumours or cells that invade neighbouring tissues and may metastasize to distant parts of the body through the lymphatic system or bloodstream.

[0397] Suitably, a cancer to be treated by a medical use or method of treatment described herein may selected from but not limited to the group consisting of: pancreatic ductal adenocarcinoma; pancreatic cancer; breast cancer; melanoma; non-small cell lung cancer; small cell lung cancer; nasopharyngeal cancer; hepatocellular cancer; colorectal cancer; oesophageal cancer; gastric cancer; anal cancer; small intestine cancer; mesothelioma; kidney cancer; renal cell carcinoma; bladder cancer; prostate cancer; ovarian cancer; vulval cancer; cervical cancer; penile cancer; uveal melanoma; retinoblastoma; sarcoma; osteosarcoma; glioblastoma; adrenocortical carcinoma; neuroblastoma; Wilms tumour; endometrial cancer; blood cancer and thyroid cancer.

[0398] In reference to cancer, the terms " treatment" and " treating" should be taken as encompassing therapy undertaken in order to prevent, slow down, or reduce an undesired physiological change or disorder, such as the growth, development or spread of cancer. Beneficial or desired results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized state of disease (which is to say, disease that is not worsening), delay or slowing of disease progression, de-staging the tumour (e.g., changing from borderline resectable to amendable for surgical resection), amelioration or palliation of the disease state, and remission (either partial or total).

[0399] Treatment may bring about prolonged survival as compared to expected survival if not receiving treatment. Alternatively, or additionally, treatment may provide a patient with an improved standard of life as compared to that which would be expected if not receiving treatment.

[0400] The medical uses and methods of treatment described herein may be of particular benefit in preventing the growth, progression or metastasis of tumours in subjects receiving treatment. In particular, the medical uses and methods of treatment described herein may be of particular benefit in preventing growth, or reducing size, of such tumours. It will be appreciated that these effects are able to beneficially reduce a subject’s tumour burden.

[0401] The medical use and methods of treating cancer provided herein may include administration of at least additional therapeutic agent. For example, the additional therapeutic agent may be an agent currently used for prevention or treatment of cancer (i.e., an anti-cancer agent). For example, if the disease is cancer the additional treatment may be selected from chemotherapy, hormone therapy, radiotherapy, immunotherapy, targeted therapy, and / or surgery.

[0402] “Chemotherapy” refers to treatment with one or more therapeutic agents to reduce or eliminate the growth or proliferation of cancer cells.

[0403] Examples of anti-cancer agents include, but are not limited to, antibodies, antibody fragments, conjugates, drugs, cytotoxic agents, proapoptotic agents, toxins, nucleases (including DNAses and RNAses), hormones, immunomodulators, chelators, boron compounds, photoactive agents or dyes, radioisotopes or radionuclides, oligonucleotides, interference RNA, peptides, anti-angiogenic agents, chemotherapeutic agents, cytokines, chemokines, prodrugs, enzymes, binding proteins or peptides or combinations thereof.

[0404] For example, chemotherapeutic drugs include vinca alkaloids, anthracyclines, epidophyllotoxins, taxanes, antimetabolites, tyrosine kinase inhibitors, alkylating agents, antibiotics, Cox-2 inhibitors, antimitotics, antiangiogenic and proapoptotic agents, doxorubicin, methotrexate, taxol, other camptothecins, and others from these and other classes of anticancer agents, and the like. Other cancer chemotherapeutic drugs include nitrogen mustards, alkyl sulfonates, nitrosoureas, triazenes, folic acid analogs, pyrimidine analogs, purine analogs, platinum coordination complexes, hormones, and the like. Suitable chemotherapeutic agents are described in REMINGTON'S PHARMACEUTICAL SCIENCES, 19th Ed. (Mack Publishing Co. 1995), and in GOODMAN AND GILMAN'S THE PHARMACOLOGICAL BASIS OF THERAPEUTICS, 7th Ed. (MacMillan Publishing Co. 1985), as well as revised editions of these publications. Other suitable chemotherapeutic agents, such as experimental drugs, are known to those of skill in the art.

[0405] Exemplary drugs include, but are not limited to, 5-fluorouracil, afatinib, aplidin, azaribine, anastrozole, anthracyclines, axitinib, AVL-101 , AVL-291 , bendamustine, bleomycin, bortezomib, bosutinib, bryostatin-1 , busulfan, calicheamycin, camptothecin, carboplatin, 10- hydroxycamptothecin, carmustine, Celebrex, chlorambucil, cisplatin (CDDP), Cox-2 inhibitors, irinotecan (CPT-1 1), SN-38, carboplatin, cladribine, camptothecans, crizotinib, cyclophosphamide, cytarabine, dacarbazine, dasatinib, dinaciclib, docetaxel, dactinomycin, daunorubicin, doxorubicin, 2-pyrrolinodoxorubicine (2P-DOX), cyano- morpholino doxorubicin, doxorubicin glucuronide, epirubicin glucuronide, erlotinib, estramustine, epidophyllotoxin, erlotinib, entinostat, estrogen receptor binding agents, etoposide (VP 16), etoposide glucuronide, etoposide phosphate, exemestane, fingolimod, floxuridine (FUdR), 3',5'-0-dioleoyl-FudR (FUdR-dO), fludarabine, flutamide, farnesyl- protein transferase inhibitors, flavopiridol, fostamatinib, ganetespib, GDC-0834, GS-1101 , gefitinib, gemcitabine, hydroxyurea, ibrutinib, idarubicin, idelalisib, ifosfamide, imatinib, L- asparaginase, lapatinib, lenolidamide, leucovorin, LFM-A13, lomustine, mechlorethamine, melphalan, mercaptopurine, 6-mercaptopurine, methotrexate, mitoxantrone, mithramycin, mitomycin, mitotane, navelbine, neratinib, nilotinib, nitrosurea, olaparib, plicomycin, procarbazine, paclitaxel, PCI-32765, pentostatin, PSI-341 , raloxifene, semustine, sorafenib, streptozocin, SU 11248, sunitinib, tamoxifen, temazolomide (an aqueous form of DTIC), transplatinum, thalidomide, thioguanine, thiotepa, teniposide, topotecan, uracil mustard, vatalanib, vinorelbine, vinblastine, vincristine, vinca alkaloids and ZD 1839.

[0406] “Radiation therapy” refers to a cancer treatment that uses high-energy x-rays or other types of radiation to kill cancer cells or keep them from growing. There are two types of radiation therapy. External radiation therapy uses a machine outside the body to send radiation toward the cancer. Certain ways of giving external radiation therapy can help keep radiation from damaging nearby healthy tissue. For example, three-dimensional conformal radiation therapy (3D-CRT) uses computers to precisely map the location of the tumour. Radiation beams are then shaped and aimed at it from several directions, which makes it less likely to damage normal tissues. Another example is, intensity-modulated radiation therapy (IMRT) is a type of 3-dimensional (3-D) radiation therapy that uses a computer to make pictures of the size and shape of the tumour. Thin beams of radiation of different intensities (strengths) are aimed at the tumour from many angles. Radiation therapy also includes proton beam radiation therapy, image guided radiation therapy (IGRT), helical-tomotherapy and photon beam radiation therapy.

[0407] Internal radiation therapy (also referred to as brachytherapy) uses a radioactive substance sealed in needles, seeds, wires, or catheters that are placed directly into or near the cancer. Internal radiation therapy may allow for higher dose of radiation in a smaller area than might be possible with external radiation treatment. Internal radiation therapy includes high-dose- rate (HDR) brachytherapy (using a highly radiative source for a relatively short e.g. 10 to 20 minute amount of time over a number of intervals) and low-dose-rate brachytherapy (use of lower doses of radiation over a longer period).

[0408] Radiation therapy may also include systemic radiation therapies such as radioimmunotherapy and peptide receptor radionuclide therapy (PRRT).

[0409] Chemotherapy and radiation therapy may both be used either sequentially and / or simultaneously. Use of both therapies or one of chemotherapy or radiation therapy may be referred to as (chemo)radiation therapy. Use of both therapies may be referred to as chemoradiation therapy.

[0410] Suitably, the cancer is a blood cancer. As used herein, the term “blood cancers” refers to a disease caused by the presence of red blood cells, white blood cells, and / or platelets with abnormal cellular proliferation. Non-limiting examples of blood cancers groups include Leukaemia, Lymphoma, Myeloma, Myelodysplastic syndromes (MDS), Myeloproliferative neoplasms (MPN), Myelodysplastic / myeloproliferative neoplasms (MDS / MPN overlap), Monoclonal gammopathy of undetermined significance (MGUS), Histiocytosis, and Mastocytosis. Non-limiting specific examples of blood cancers include Acute erythroid leukaemia, Acute lymphoblastic leukaemia (ALL), Acute megakaryoblastic leukaemia, Acute myeloid leukaemia (AML), Acute promyelocytic leukaemia (APL), Chronic lymphocytic leukaemia (CLL), Chronic myeloid leukaemia (CML), Chronic myelomonocytic leukaemia (CMML), Childhood leukaemia, Hairy cell leukaemia (HCL), Large granular lymphocytic leukaemia (LGLL), Mast cell leukaemia (MCL), Burkitt lymphoma, Central nervous system (CNS) lymphoma, Diffuse large B-cell lymphoma (DLBCL), Double-hit lymphoma, Follicular lymphoma, Grey zone lymphoma, High-grade B- cell lymphoma not otherwise specified (NOS), Hodgkin lymphoma, MALT lymphoma, Mantle cell lymphoma, Nodal marginal zone lymphoma, Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), Non-Hodgkin lymphoma, Peripheral T cell lymphoma (PTCL), Skin lymphoma (cutaneous lymphoma), Small lymphocytic lymphoma (SLL), Splenic marginal zone lymphoma, Triple-hit lymphoma, Waldenstrom macroglobulinaemia (WM), Polycythaemia vera (PV), Essential thrombocythaemia (ET), Myelofibrosis, Erdheim-Chester disease (ECD), Langerhans cell histiocytosis, and Mast cell leukaemia (MCL).

[0411] Suitably, the additional therapeutic is a immune checkpoint inhibitor. As used herein, an “immune checkpoint inhibitor” means an agent that inhibits proteins or peptides (e.g. immune checkpoint proteins) which are blocking the immune system, e.g., from attacking cancer cells. Suitably, the immune checkpoint protein blocking the immune system prevents the production and / or activation of T cells. An immune checkpoint inhibitor can be an antibody or antigenbinding fragment thereof, a protein, a peptide, a small molecule, or combination thereof. Typically, the inhibitor interacts directly to a target immune checkpoint protein (or its ligand, where appropriate) and thereby disrupts its function / biological activity. For example, it may bind directly to a target immune checkpoint protein (or its ligand, where appropriate). In one example, direct binding to a target immune checkpoint protein (or its ligand, where appropriate) inhibits, prevents or reduces the formation of protein complexes which are needed for immune checkpoint protein function / biological activity.

[0412] A review describing immune checkpoint pathways and the blockade of such pathways with immune checkpoint inhibitor compounds is provided by Pardoll in Nature Reviews Cancer (April, 2012), pages 252-264. Immune check point inhibitor compounds display anti-tumor activity by blocking one or more of the endogenous immune checkpoint pathways that downregulate an anti-tumor immune response. The inhibition or blockade of an immune checkpoint pathway typically involves inhibiting a checkpoint receptor and ligand interaction with an immune checkpoint inhibitor compound to reduce or eliminate the signal and resulting diminishment of the anti-tumor response.

[0413] The immune checkpoint inhibitor compound may inhibit the signalling interaction between an immune checkpoint receptor and the corresponding ligand of the immune checkpoint receptor. The immune checkpoint inhibitor compound can act by blocking activation of the immune checkpoint pathway by inhibition (antagonism) of an immune checkpoint receptor (some examples of receptors include CTLA-4, PD-1 , and NKG2A) or by inhibition of a ligand of an immune checkpoint receptor (some examples of ligands include PD-L1 and PD-L2). In such examples, the effect of the immune checkpoint inhibitor compound is to reduce or eliminate down regulation of certain aspects of the immune system anti-tumor response in the tumor microenvironment.

[0414] Suitably, the immune checkpoint inhibitor inhibits the CTLA-4 pathway or the PD-L1 / PD-1 pathway (examples thereof are provided in, e.g., WO 2016 / 062722).

[0415] Suitably, the immune checkpoint inhibitor is an anti-CTLA-4 antibody or derivative or antigenbinding fragment thereof. Examples of anti-CTLA-4 antibodies and derivatives and fragments thereof are described in, e.g., US 6,682,736; US 7,109,003; US 7,123,281 ; US 7,411 ,057; US 7,807,797; US 7,824,679; US 8,143,379; US 8,491 ,895, and US 2007 / 0243184. In some examples, the anti-CTLA-4 antibody is tremelimumab or ipilimumab.

[0416] Suitably, the immune checkpoint inhibitor is an anti-PD-L1 antibody or derivative or antigenbinding fragment thereof. In some examples, the anti-PD-L1 antibody or derivative or antigenbinding fragment thereof selectively binds a PD-L1 protein or fragment thereof. Examples of anti-PD-L1 antibodies and derivatives and fragments thereof are described in, e.g., WO 01 / 14556, WO 2007 / 005874, WO 2009 / 089149, WO 2011 / 066389, WO 2012 / 145493; US 8,217,149, US 8,779,108; US 2012 / 0039906, US 2013 / 0034559, US 2014 / 0044738, and US 2014 / 0356353. In some embodiments, the anti-PD-L1 antibody is MEDI4736 (durvalumab), MDPL3280A, 2.7A4, AMP-814, MDX-1 105, atezolizumab (MPDL3280A), or BMS-936559.

[0417] Suitably, the immune checkpoint inhibitor is an anti-PD-1 antibody or derivative or antigenbinding fragment thereof. Suitably, the anti-PD-1 antibody selectively binds a PD-1 protein or fragment thereof. Suitably, the anti-PD-1 antibody is nivolumab, pembrolizumab, or pidilizumab.

[0418] Suitably, the immune checkpoint inhibitor is an anti-NKG2A compound, such as an anti- NKG2A antibody. Examples of anti-NKG2A antibodies and derivatives and fragments thereof are described in WO 2016 / 041947, the content of which is hereby incorporated by reference in its entirety including, but not limited to, the sequence listings.

[0419] Suitably, the immune checkpoint inhibitor compound is a small organic molecule (molecular weight less than 1000 daltons), a peptide, a polypeptide, a protein, an antibody, an antibody fragment, or an antibody derivative. In some embodiments, the immune checkpoint inhibitor compound is an antibody. In some embodiments, the antibody is a monoclonal antibody, specifically a human or a humanized monoclonal antibody.

[0420] Methods for the preparation and use of immune checkpoint antibodies are described in the following illustrative publications. The preparation and therapeutic uses of anti-CTLA-4 antibodies are described in U.S. Patent Nos. 7229628 (Allison), 7311910 (Linsley), and 8017144 (Korman). The preparation and therapeutic uses of anti-PD-1 antibodies are described in U.S. Patent No. 8008449 (Korman) and U.S. Patent Application No. 201 1 / 0271358 (Freeman). The preparation and therapeutic uses of anti-PD-L1 antibodies are described in U.S. Patent No. 7943743 (Korman). The preparation and therapeutic uses of anti- TIM-3 antibodies are described in U.S. Patent Nos. 8101176 (Kuchroo) and 8552156 (Tagayanagi). The preparation and therapeutic uses of anti-LAG-3 antibodies are described in U.S. Patent Application No. 2011 / 0150892 (Thudium) and International Publication Number W02014 / 008218 (Lonberg). The preparation and therapeutic uses of anti-KIR antibodies are described in U.S. Patent No. 81 19775 (Moretta). The preparation of antibodies that block BTLA regulated inhibitory pathways (anti-BTLA antibodies) are described in U.S. Patent No. 8563694 (Mataraza).

[0421] Immune checkpoint inhibitors that may be administered to a subject include but are not limited to an anti-PD-1 antibody, anti-PD-L1 antibody, anti-LAG-3 antibody, anti-TIGIT antibody, anti- KLRB1 antibody, anti-LILRB2 antibody, anti-LILRB4 antibody, anti-LILRB2 and LILRB4 antibody and / or anti-TIM-3 antibody. Examples of immune checkpoint inhibitors include atezolizumab, ipimilumab, pembrolizumab, lambrolizumab (MK-3475, MERCK), nivolumab (BMS-936558, BRISTOL-MYERS SQUIBB), AMP-224 (MERCK), pidilizumab (CT-01 1 , CURETECH LTD) and tislelizumab. Exemplary anti-PD-L1 antibodies include MDX-1105 (MEDAREX), MEDI4736 (MEDIMMUNE) MPDL3280A (GENENTECH) and BMS-936559 (BRISTOL-MYERS SQUIBB). Other examples include LILRB2 and LILRB4 antibodies described in US20190194327A1 .

[0422] The inhibitor need not be an antibody, but can be a small molecule or other agent or compound. If the inhibitor is an antibody it may be a polyclonal, monoclonal, fragment, single chain, or other antibody variant construct. Inhibitors may target any immune checkpoint protein known in the art, including but not limited to, CTLA-4, PDL1 , PDL2, PD-1 , B7-H3, B7-H4, BTLA, HVEM, TIM3, GAL9, LAG3, VISTA, KIR, 2B4, CD160, CGEN-15049, CHK1 , CHK2, A2aR, and the B-7 family of ligands. Combinations of inhibitors for a single target immune checkpoint or different inhibitors for different immune checkpoints may be used. In particular the immune checkpoint therapy may be an inhibitor of one or more of CD274 (PD-L1), PDCD1 LG2 (PD-L2), TIGIT, HAVCR2 (TIM-3), LAG-3, KLRB1 , LILRB2 and / or LILRB4. For example, the immune checkpoint inhibitor may be selected from one or more of Nivolumab, Atezolizumab, avelumab, durvalumab , and ipilimumab.

[0423] Suitably, the additional therapeutic is a chimeric antigen receptor therapeutic. Chimeric antigen receptor therapeutics typically include chimeric antigen receptor cells, which may be chimeric antigen receptor T cells, chimeric antigen receptor NK cells, and the like. Examples of CAR therapeutics Abecma®, Breyanzi ®, Kymriah ®, Tecartus ®, Yescarta ®, and Carvykti ®. Other examples of CAR therapeutics can be found in, for example, WO2019220109A1 , US11034750B2, WO2013123061 A1 , US20130287748A1 , WO2014055668A1 ,

[0424] WO2014138704A1 , WO2015075468A1 , and WO2017216561 A1 .

[0425] Transplantation

[0426] The multivalent proteins, multival...

Claims

CLAIMS1 . A multivalent protein comprising: a. a first region capable of binding to one or more effector proteins, the effector protein comprising or associated with protein kinase activity; and b. a second region capable of binding to one or more target proteins, wherein the target protein signals through a phosphorylation mechanism.

2. The multivalent protein of claim 1 , wherein the first and second regions are operably linked.

3. The multivalent protein of any preceding claim, wherein the multivalent protein comprises a linker interposed between the first and second regions.

4. The multivalent protein of any preceding claim, wherein at least one of the first and / or second region each comprise a binding domain for binding to the effector and / or target proteins.

5. The multivalent protein of claim 4, wherein each binding domain comprises an antigen-binding domain or a protein-binding ligand, optionally wherein: a. the antigen-binding domain comprises an antigen-binding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VHdomain, a VLdomain, a single domain antibody (dAb), a VNAR domain, a VHH domain, a T cell receptor (TCR) binding subunit, a TCR-like polypeptide, an antibody mimetic such as a DARPin, anticalin, affibody, minibinder, cyclic or bicyclic peptide or a functional fragment of any thereof ; and / or b. the protein-binding ligand comprises a cytokine, a growth factor, a receptor extracellular domain (ECD), a peptidic hormone, GPCR ligand, or a functional variant of any thereof.

6. The multivalent protein of any preceding claim, wherein the multivalent protein comprises at least one first polypeptide chain comprising at least a portion of the first and / or second region and at least one second polypeptide chain comprising at least a second portion of the first and / or second region.

7. The multivalent protein of any preceding claim, wherein the first region and / or the second region or a portion thereof each comprise at least one Fc region; optionally wherein at least a portion of the first region and at least a portion of the second region are linked via the Fc regions or a portion thereof wherein the first region and / or the second region or a portion thereof each comprise at least a portion of a TCR constant domain.

8. The multivalent protein of any of claims 1 to 6, wherein the multivalent protein comprises a multivalent antibody structure; optionally wherein the multivalent antibody structure is selected from a single domain bispecific antibody, diabody, Dual-Affinity Re-Targeting molecule (DART), Tandem ScFv (BiTE), CrossMab, CrossMab-Fab, tandem CrossFab, TCR-like antibody, and tandem diabody or variants thereof or the multivalent protein comprises a multivalent non-antibody based scaffold protein.

9. The multivalent protein of any preceding claim, wherein the effector protein and / or target protein each comprise one or more extracellular domains, optionally wherein the effector protein and / or target protein each comprise one or more cell surface receptor proteins.

10. The multivalent protein of any preceding claim, wherein the effector protein and target protein are present on the same cell; optionally wherein the multivalent protein binds the effector and target protein in cis.11 . The multivalent protein of any preceding claim, wherein the effector protein comprises: a. a receptor-protein tyrosine kinase; or b. a protein that comprises one or more extracellular domains that interact with one or more intracellular protein kinases; optionally wherein the effector protein is selected from CD8, CD4, CD48, CD2, CD59, CD55, CD24, and CD14.

12. The multivalent protein of claim 11 , wherein the one or more intracellular protein kinases comprises one or more Src family kinases; optionally wherein the one or more Src family kinases comprise Src, Yes, Fyn, Lyn, Hck, Lek, Fgr, Blk, and / or Yrk.

13. The multivalent protein of any preceding claim, wherein the target protein mediates signalling through a tyrosine motif; optionally wherein the tyrosine motif is selected from one or more of an ITAM, ITSM, or ITIM motif or a related intracellular motif that is chemically modified by a phosphorylation of a tyrosine residue.

14. The multivalent protein of any preceding claim, wherein the target protein comprises: a. an immune-checkpoint receptor; b. a cytokine receptor; c. an interferon receptor; d. tumour necrosis factor receptor e. a growth factor receptor; optionally wherein the target protein comprises an inhibitory receptor protein; further optionally wherein the inhibitory receptor protein is selected from PD-1 , BTLA, CD5, CD6, CD200R, TIM-3, TIGIT, CD22, Siglec-7, Siglec-14, Siglec-9, Siglec-6, CD300a, FcyRIIB, NKG2A, CD72, CTLA-4, LAIR1 , ILT-2, ILT-4, CD31 , SIRPcx, members of the CEACAM family, members of the SLAM family of surface receptors, and members of the KIR family of surface receptors.

15. The multivalent protein of any preceding claim, wherein the multivalent protein comprises from N-terminal to C-terminal a. Domain A comprising a binding region for binding to the effector protein; a linker; andDomain B comprising a binding region for binding to the target protein; or b. Domain A comprising a binding region for binding to the target protein;a linker; andDomain B comprising a binding region for binding to the effector protein; or c. Domain A comprising a first portion of a binding region for binding to the effector protein and a first portion of a binding region for binding to the target protein; a linker;Domain B comprising a second portion of the binding region for binding to the effector protein and a second portion of the binding region for binding to the target protein.

16. The multivalent protein of any preceding claim, wherein the multivalent protein comprises a first region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 2, 14, 15, 20, 21 , 22, 23, 24, 49, 50, 51 , 52 and a second region comprising an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1 , 5, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, or 48.

17. The multivalent protein of any preceding claim, wherein the multivalent protein comprises an amino acid sequence having at least 80% sequence identity to anyone of SEQ ID NOs: 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 122, 123, 124, or 125.

18. The multivalent protein according to any one of claims 1 to 17, wherein the multivalent protein is a multivalent antibody.

19. The multivalent protein according to claim 18, wherein the multivalent antibody is a bispecific antibody, or antigen-binding fragment thereof.

20. A nucleic acid composition comprising one or more nucleic acid molecules each encoding at least a portion of a multivalent protein according to any one of claims 1 to 19; optionally wherein the nucleic acid molecules are comprised within one or more expression cassettes or vectors.21 . A recombinant cell comprising the nucleic acid composition according to claim 20.

22. A method of producing a multivalent protein according to any one of claims 1 to 19, the method comprising: providing a recombinant cell according to claim 21 ; and expressing the one or more nucleic acid molecules to produce the multivalent protein.

23. A pharmaceutical composition comprising a multivalent protein according to any one of claims 1 to 19, a nucleic acid composition according to claim 20, or a recombinant cell according to claim 21 .

24. A method of treating a disease or condition in a subject, the method comprising administering an effective amount of a multivalent protein according to any one ofclaims 1 to 20, a nucleic acid composition according to claim 20, a recombinant cell according to claim 21 , or a pharmaceutical composition according to claim 23, to a subject in need thereof.

25. A method for modulating cell signalling mediated by and a target protein, wherein the target protein signals through a phosphorylation mechanism in a subject, the method comprising administering an effective amount of a multivalent protein according to any one of claims 1 to 19, a nucleic acid composition according to claim 20, a recombinant cell according to claim 21 , or a pharmaceutical composition according to claim 23, to a subject in need thereof.

26. A multivalent protein according to any one of claims 1 to 19, a nucleic acid composition according to claim 20, a recombinant cell according to claim 21 , or a pharmaceutical composition according to claim 23, for use as a medicament.

27. A multivalent protein according to any one of claims 1 to 19, a nucleic acid composition according to claim 20, a recombinant cell according to claim 21 , or a pharmaceutical composition according to claim 23, for use in treating : an autoimmune disease; inflammation; allergies; transplantation; or cancer.

28. The method according to claims 24 or 25 or the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition for use according to claim 26 or 27, wherein the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition modulates an immune response in the subject.

29. The method according to any one of claims 24, 25, and 28, or the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition for use according to any one of claims 26 to 28, wherein the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition modulates inflammation in the subject.

30. The method according to any one of claims 24, 25, 28 and 29 or the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition for use according to any one of claims 26 to 29, wherein the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition suppress T cell activation in the subject.31 . The method according to any one of claims 24, 25 and 28 to 30 or the multivalent protein, nucleic acid composition, recombinant cell, multivalent antibody or pharmaceutical composition for use according to any one of claims 26 to 30, wherein the multivalent protein, nucleic acid composition, recombinant cell or pharmaceutical composition enhance inhibitory signalling of checkpoint proteins in the subject.

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