Agents, methods and uses thereof

Abstract

The present invention relates to methods and conjugates to improve the penetrability of biological substances into the tumour microenvironment (TME) for therapeutic purposes, suitably the agent comprises at least: i a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain.

Description

[0001] AGENTS, METHODS AND USES THEREOF

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to methods and conjugates to improve the penetrability of biological substances into the tumour microenvironment (TME) for therapeutic purposes.

[0004] BACKGROUND OF THE INVENTION

[0005] Cancers are a major contributor to disease burden worldwide, and projections forecast that global cancer burden will continue to grow. Cancer is a generic term for a large group of diseases that can affect any part of the body. A defining feature of cancer is the rapid creation of unwanted cells that grow beyond their usual boundaries, and which can then invade adjoining parts of the body and spread to other organs.

[0006] Antibody-based therapies have revolutionised therapies, particularly in the field of oncology. Engineered antigen-binding domains can bind to specific tumour markers and can activate or inactivate signalling pathways. This results in the slowing down or stopping of cell proliferation and tumour growth, or the shrinkage of tumours (Zahavi D, Weiner L (2020), Monoclonal Antibodies in Cancer Therapy, Antibodies (Basel), 9(3);34). Engineered antigen-binding domains are also used in immunotherapy, where they can activate or deactivate immune cells within the tumour to stop or slow down tumour proliferation and growth.

[0007] The success of these antibody-based therapies is due to the high specificity of the antigenbinding domains to their oncological targets. This mitigates any non-specific binding to similar antigens giving rise to better targeting. Antibody-based therapies can be multivalent, so a single antibody can engage its target multiple times which gives rise to additional efficacy that small molecules do not give (Imai K, Takaoka A (2006), Comparing antibody and smallmolecule therapies for cancer, Nat Rev Cancer, 6(9);714-27).

[0008] Antibodies also contain a fragment crystallisable (Fc) region. This region has several functions, one of which is to extend the antibody's half-life. This is in part achieved by the Fc region increasing the size of the antibody taking it past the glomerular filtration barrier (GFB) threshold.

[0009] A main problem in the field remains the penetration of the antibody-based therapies into the TME. It is estimated that only 0.001-7% of the injected antibody makes it into the heart of the tumour (Khongorzul et al., (2020), Antibody-Drug Conjugates: A Comprehensive Review, Mol Cancer Res, 18(1);3-19). A lower penetration of a biologic into the TME is associated with a sub-optimal efficacy of the biologic. This sub-optimal efficacy leads to the use of higher doses of the biologic being administered to achieve a therapeutic effect; however, higher doses can be detrimental and / or toxic to the patient.

[0010] A contributing factor of the lower penetration is thought to be the large sizes of antibodies and thus, much effort has been focused on generating small antibody fragments for better efficacy. Modelling experiments also suggest that smaller antibody fragments have better penetration into tissues (Thurber et al., (2008), Antibody tumour penetration: transport opposed by systemic and antigen-mediated clearance, Adv Drug Deliv Rev, 60(12); 1421-34).

[0011] The small antibody fragments often a lack of an Fc region, which lowers the half-life (Li et al., 2019) of the antibody and results in a lower efficacy.

[0012] In heathy vascularised tissue, cells are only a few micrometres (pm) away from a blood vessel, so organ / cell penetration is not of concern. However, the large cell mass acquired in tumour formation causes an increase in the distance between the nearest blood vessel and the cells. This makes accessibility and delivery of therapies to the cells an issue.

[0013] This also gives marked characteristics within the tumour that are unusual. For example, the distance from blood vessels leads to hypoxia within the tumour; low concentrations of oxygen and elevated levels of carbon dioxide, resulting in lower pH levels. In addition, the environment promotes carcinogenesis. To achieve this, specific proteases are expressed which allow for rogue cells to escape into the blood stream, invade local tissues and eventually metastasise. Thus, during tumour development, aberrantly expressed proteases are found in the TME. The expression of different protease combinations varies depending on the tumour type and location. Such proteases have been used to specifically target antibody activity in vivo for example WO2013192546A1 and WO2015066279A2.

[0014] Some protease cleavage sites are well known in the scientific literature, and cleavable domains comprising such cleavage sites can be readily constructed using established genetic engineering techniques and / or by chemical synthesis techniques known in the art, while other protease cleavage sites are not well known so designing such cleavable domains requires more experimentation.

[0015] Although these have been used to improve specificity of antibodies, the problem remains that the antibodies have poor penetration and thus poor efficacy. The present invention seeks to solve this problem. SUMMARY OF THE INVENTION

[0016] The invention is based on the use of tumour-specific proteases to enhance antibody penetration by specific cleavage of the antibody at specific engineered sites when the antibody has reached the TME. The invention is based on the discovery that such sequences around the hinge region of antibodies does not affect the expression or function of the antibody.

[0017] The invention is also based on cleavage of the therapeutic agent and thus reduction of its size at the tumour or in the vicinity of the unwanted cells or the tumour, significantly improving specificity, penetrability and therefore efficacy of the therapeutic agent.

[0018] Accordingly, in one aspect, the invention provides an engineered agent comprising at least: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain.

[0019] The invention provides introduction of a cleavable domain between a therapeutic domain and a stabilisation domain, such that when the cleavage site is selectively cleaved in the vicinity of the unwanted cells, the therapeutic domain can be released from a stabilisation domain in the vicinity of the unwanted cells and can bind to an antigen on or in the unwanted cells.

[0020] The invention provides means to improve the penetrability of an agent into a tumour by using size and diffusion to penetrate deeper into the hypoxic parts of the tumour to achieve enhanced agent efficacy.

[0021] BRIEF DESCRIPTION OF THE FIGURES

[0022] Figure 1 - Illustrative schematic of where the therapeutic domain is Fab and the cleavage site is within the upper or lower the hinge region of an antibody. Upon entering the TME, conditions allow for the cleavage of the antibody into 3 domains, the Fab domains (x2) and Fc domain (xl).

[0023] Figure 2 - Illustrative schematic of where the therapeutic domain is Fab and the cleavable domain is present after the hinge region of an antibody. Upon entering the TME, conditions allow for the cleavage of the antibody into 2 domains, the F(ab')2 domain (xl) and Fc domain (xl).

[0024] Figure 3 - Illustrative schematic of where the therapeutic domain is not Fab and the cleavable domain is present before the hinge region of an Fc containing entity. Upon entering the TME, conditions allow for the cleavage of the agent into 3 domains, the therapeutic domains (x2) and Fc domain (xl). Figure 4 - Illustrative schematic of where the therapeutic domain is not Fab and the cleavable domain is present after the hinge region of an Fc containing entity. Upon entering the TME, conditions allow for the cleavage of the agent into 2 domains, the therapeutic domain (xl) and Fc domain (xl).

[0025] Figure 5 - Example of where the cleavable domain is present at a linker domain that separates the therapeutic domain from the stabilisation domain of a biologic.

[0026] Figure 6 - Illustrative schematic of different positions of the cleavable domain within the hinge region. (A) D1-D4 designs - the cleavable site is within the upper hinge region; (B) D6-D7 designs - the cleavable site is within the lower hinge region; (C) D5 design was engineered with a glycosylation site and gives a negative control. The position and length of the linker domains differ from design to design. The cleavable site may also be within the middle hinge (not shown).

[0027] Figure 7 - Assessment of antibody concentration by Protein A Octet.

[0028] Figure 8 - Gel image showing Protein Quality Analysis (SDS-PAGE).

[0029] Figure 9 - Repeat assessment of antibody concentration by Protein A Octet.

[0030] Figure 10 - Repeat gel image showing Protein Quality Analysis (SDS-PAGE). The box shows the antibody heavy chains (~50kDa).

[0031] Figure 11 - Chromatographic assessment of candidate expression and purification. (A) trastuzumab (WT also known as mAb) Control and (B) DI.

[0032] Figure 12 - Gel image showing (A) Non-Reduced Protein Quality Analysis (SDS-PAGE) and (B) Reduced Protein Quality Analysis (SDS-PAGE). Lanes: 1 = Molecular Weight Marker in kilodaltons (KDa); 2 = Trastuzumab mAb; 3 = Trastuzumab DI; 4 = Trastuzumab D2; 5 = Trastuzumab D3; 6 = Trastuzumab D4; 7 = Trastuzumab D5; 8 = Trastuzumab D6; 9 = Trastuzumab D7.

[0033] Figure 13 - Binding of trastuzumab mAb and engineered designs to hHER2 (human HER2).

[0034] Figure 14 - 12-point dilution kinetics measurement. (A) trastuzumab mAb and (B) DI.

[0035] Figure 15 - Gel image showing liberation of F(ab) for D1-D5 and liberation of F(ab')2 for D6-

[0036] D7 fragments from control mAb, DI and D2 antibodies by uPA Protease. 1 = Molecular Weight Marker; 2 = mAb + 0 nM Protease; 3 = mAb + 23.5 nM Protease; 4 = mAb + 235 nM Protease; 5 = DI + 0 nM Protease; 6 = DI + 23.5 nM Protease; 7 = DI + 235 nM Protease; 8 = D2 0 nM Protease; 9 = D2 + 23.5 nM Protease; 10 = D2 + 235 nM Protease; 11 = D3 + 0 nM Protease; 12 = D3 + 23.5 nM Protease; 13 = mAb + 235 nM Protease; 14 = D4 + 0 nM Protease; 15 = D4 + 23.5 nM Protease; 16 = D4 + 235 nM Protease; 17 = D5 + 0 nM Protease; 18 = D5 + 23.5 nM Protease; 19 = D5 + 235 nM Protease; 20 = D6 + 0 nM Protease; 21 = D6 + 23.5 nM Protease; 22 = D6 + 235 nM Protease; 23 = D7 + 0 nM Protease; 24 = D7 + 23.5 nM Protease; 25 = D7 + 235 nM Protease. Fc is the middle row of bands, and F(ab) and F(ab')2 + / Light chain is the lower row of bands. F(ab) and F(ab')2 appear as the same size on the gel because DTT reduces the liberated F(ab')2 into 2 Fabs.

[0037] Figure 16 - Binding of trastuzumab and engineered antibodies to hHER2 after uPA treatment - Fab detection.

[0038] Figure 17 - Binding of Trastuzumab and engineered antibodies to FC Gamma Receptors. (A) FC Gamma Receptor la; (B) FC Gamma Receptor Ila; (C) FC Gamma Receptor lib; (D) FC Gamma Receptor Illa.

[0039] Figure 18 - Larger Scale 20mg Expression of (A) Trastuzumab mAb 98.5% monomer and (B) Trastuzumab D3 94.3% monomer.

[0040] Figure 19 - SDS PAGE gel showing expression and purity of Trastuzumab-mAb and D3. 1 = Trastuzumab-mAb and 2 = Trastuzumab D3. NR = non-reduced and R = reduced. A PageRuler Plus prestained ladder was used.

[0041] Figure 20 - Binding of Larger Scale Preps of trastuzumab mAb and D3 to hHER2 by ELISA.

[0042] Figure 21 - Expression of Trastuzumab-mAb and Trastuzumab DI in CHO-K1 Cell Lines.

[0043] Figure 22 - Comparison of expressions from CHO-K1 and Expi-CHO.

[0044] Figure 23 - SDS-PAGE gel showing expression and purity of trastuzumab engineered antibodies with different cleavage sites 1. Molecular Weight Marker, 2. Empty Lane, 3. Trastuzumab D9 (with ADAM10 site)

[0045] Figure 24 - Binding of Trastuzumab mAb and Trastuzumab D3 and D9 to hHER2.

[0046] Figure 25 - Non reduced SDS-PAGE gel showing the expression and purity of 1. Molecular Weight Marker, 2. Bevacizumab mAb, 3. Bevacizumab D13, 4. Cetuximab mAb, 5. Cetuximab D12, 6. M5A mAb, 7. M5A D15, 8. Pembrolizumab mAb, 9. Pembrolizumab D14a, 10. Pembrolizumab D14b, 11., Ipilimumab mAb, 12. Ipilimumab D16.

[0047] Figure 26 - (A) Binding of Cetuximab mAb and Cetuximab D12 to hEGFR; (B) Binding of Bevacizumab mAb and Bevacizumab D13 to hVEGF.

[0048] Figure 27 - SDS-PAGE gel showing protease digestion of (A) 1. Molecular Weight Marker, 2. Bevacizumab mAb + 0 nM Protease, 3. Bevacizumab mAb + 23.5 nM Protease, 4. Bevacizumab mAb + 235 nM Protease, 5. Bevacizumab D13 + 0 nM Protease, 6. Bevacizumab D13 + 23.5 nM Protease, 7. Bevacizumab D13 + 235 nM Protease, 8. Cetuximab mAb + OnM Protease, 9. Cetuximab mAb + 23.5 nM Protease, 10. Cetuximab mAb + 235 nM Protease, 11. Cetuximab D12 + 0 nM Protease, 12. Cetuximab D12 + 23.5 nM Protease, 13. Cetuximab D12 + 235 nM Protease; (B) 1. Molecular Weight Marker, 2. M5A mAb + 0 nM Protease, 3. M5A mAb + 23.5 nM Protease, 4. M5A mAb + 235nM Protease, 5. M5A D15 + OnM Protease, 6. M5A D15 + 23.5nM Protease, 7. M5A D15 + 235nM Protease (C) 1. Molecular Weight Marker, 2. Pembrolizumab mAb, 3. Pembrolizumab D14a + 500 pM Protease, 4. Pembrolizumab D14b + 500 pM; (D) 1. Molecular Weight Marker, 2. Trastuzumab D3 3. Trastuzumab D9. Protease was added at a 1 : 1 molar concentration of antibody to protease.

[0049] Figure 28 - Binding of different antibody-derived fragments to their respective targets (A) Pembrolizumab, D14a and D14b to hPDl-HIS; (B) M5A and D15 to hCEACAM-5; (C) Ipililmumab and D16 to hCTLA-4. Insertion of the cleavage side does not significantly affect the ability of the engineered antibody to bind to its target.

[0050] Figure 29 - Illustrative schematic of different types of experimentally-tested therapeutic domains. (A) An agent wherein the therapeutic domain is Fab; (B) An agent wherein the therapeutic domain is VHH; (C) An agent wherein the therapeutic domain is ScFv. In (A), (B) and (C) the cleavable domain is located in the upper hinge and the stabilisation domain is an Fc region.

[0051] Figure 30 - SDS PAGE gel showing (A) expression and purity of various engineered agents comprising VHH or ScFv as a therapeutic domain. Lanes: 1 = Molecular Weight Marker; 2 = anti-EGFR VHH-FC (D17); 3 = anti-EGFR VHH Control (7D12-FC); 4 = anti-HER2 ScFv-FC (D18); 5 = anti-HER2 ScFv-FC control (Trastuzumab Vl-Vh long linker); (B) protease digestion of VHH and ScFv-FC constructs with uPA. Lanes: 1. Molecular Weight Marker, Lane 2. D17 with 250 pM uPA; 3 = anti-EGFR VHH Control with 250 pM uPA; 4 = D18 with 250 pM uPA; 5 = anti- HER2 ScFv-FC control with 250 pM uPA. All proteases were added with a 1: 1 molar concentration of antibody to protease; (C) Assessment of binding of anti-EGFR VHH-FC (D17) to hEGFR by ELISA; (D) Assessment of binding of anti-HER2 ScFv-FC (D18) to hHER2 by ELISA. Figure 31 - Binding of tumour samples to human HER.2 with Vk light chain detection. (A) In a mouse study, 5 "Group 2" (G2) mice were injected with Trastuzumab mAb and 5 "Group 3" (G3) mice were injected with Trastuzumab D3. G3 mice showed an increased accumulation of Trastuzumab D3 in the tumour in comparison to G2 mice's accumulation of Trastuzumab mAb. (B) Amalgamation of data from (A) mouse study showing that there was 15.6 pM Trastuzumab D3 in G3 mice and 7.8 pM Trastuzumab mAb in G2.

[0052] DETAILED DESCRIPTION OF THE INVENTION

[0053] The agent may be a therapeutic agent. By a "therapeutic agent", we include the meaning of it having a beneficial or desired result including and preferably a beneficial or desired clinical result, i.e. a treatment (curative) agent or a "prophylactic" agent administered to treat and / or prevent disease. By agent, we include the meaning of biological agents which include proteins, oligopeptides, polypeptides, enzymes, antibodies and parts thereof, vaccines, nucleotides and the like, antibody analogues, antibody mimetics, immunoglobulins, immunomodulators, blood, blood components, cells, allergens, genes, viruses, toxins, venoms or combinations thereof.

[0054] In one embodiment, the agent is an engineered agent. By "engineered agent" we include the meaning that the agent is not a naturally occurring agent and may be synthesised. In one embodiment, the stabilisation domain and therapeutic domain are naturally occurring together (such as in an antibody); whereas the cleavable domain is engineered into the hinge region.

[0055] By 'therapeutic domain', we include the meaning of any domain or entity which has therapeutically beneficial effect on a subject. The therapeutic domain may bind to an endogenous target, for example may bind a tumour antigen that is endogenous to the subject.

[0056] By 'cleavable domain', we include the meaning of any cleavable domain or entity which is susceptible to cleavage at the tumour by another domain or entity. Cleavage can occur at one or more locations within the cleavable domain. Such a site within the cleavable domain is known as cleavage site. Preferably, the cleavable domain is a peptide that includes a substrate for an enzyme. In one embodiment, the cleavable domain comprises only the cleavage site.

[0057] The skilled person will understand that the cleavable domain may overlap with several residues of the therapeutic domain and / or the stabilisation domain so long as when the cleavage site is positioned within these residues (via means elsewhere described herein), the therapeutic domain and / or the stabilisation domain maintain their functions as elsewhere described herein, and in particular the therapeutic domain maintains its therapeutic and / or binding activity after cleavage as described elsewhere herein. In a particular embodiment, any overlap is preferably at the C terminus of the therapeutic domain and / or at the N-terminus of the stabilisation domain. In antibodies, there are naturally occurring cleavage sites for non-tumour specific proteases and or bacterial enzymes (Brezski and Jordan 2010, Cleavage of IgGs by proteases associated with invasive diseases, mAbs 2:3, 212-220). In one embodiment the agent of the invention does not include a naturally occurring cleavage site. Accordingly, in one embodiment the agent of the invention does not comprise any cleavable domains that are susceptible to following enzymes: papain, pepsin, glutamyl endopeptidase I (GluV8), immunoglobulin-degrading enzyme of Streptococcus pyogenes (IdeS), Streptopain (SpeB), pseudolysin, mirabilysin, trepolisin.

[0058] In one embodiment, the cleavable domain is artificially introduced into a naturally occurring agent. By "the cleavable domain is artificially introduced" we include the meaning that the cleavable domain is not found in the naturally occurring agent. The agent is engineered such that a cleavable domain is artificially introduced. In one embodiment, the cleavable domain is an engineered cleavable domain. In one embodiment, part or all of the cleavable domain has been engineered into an antibody.

[0059] As used herein, the term "antibody" includes but is not limited to polyclonal, monoclonal, chimeric, single chain, Fab fragments, fragments produced by a Fab expression library and multispecific (e.g. bispecific) antibodies. Such fragments include fragments of whole antibodies which retain their binding activity for a target substance, Fv, F(ab') and F(ab')2 fragments, as well as single chain antibodies (scFv), fusion proteins and other synthetic proteins which comprise the antigen-binding site of the antibody. A targeting moiety comprising only part of an antibody may be advantageous by virtue of optimising the rate of clearance from the blood and may be less likely to undergo non-specific binding due to the Fc region. Also included are domain antibodies (dAbs), diabodies, nanobodies (such as camelid antibodies, engineered camelid antibodies, shark antibodies or llama antibodies). The advantages of using antibody fragments, rather than whole antibodies, are several-fold. The smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue. Moreover, antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli or yeast, thus allowing convenient production in the laboratory and economical production on a commercial scale.

[0060] The antibody may be of any of the IgG, IgE, IgA, IgM and IgD classes and may be derived from any species. If the antibody is an IgG, it may be any of IgGl, IgG2, IgG3 or IgG4. It is preferred, however, that when the agent is for administration to a particular host, that the antibody, or at least the constant regions thereof, are derived from that host. The antibodies may be human antibodies in the sense that they have the amino acid sequence of human antibodies with specificity for the selected antigen. Alternatively, they may be mouse, chimeric or humanized antibodies. For example, when the agent is to be administered to a human, the antibody is preferably a human antibody or a humanized antibody, and so on.

[0061] Suitable antibodies that bind to particular antigens expressed by unwanted cells can be made by the skilled person using technology long-established in the art. Methods of preparation of monoclonal antibodies and antibody fragments are well known in the art and include hybridoma technology.

[0062] By 'stabilisation domain', we include the meaning of any domain or entity that is able to stabilise or extend the half-life of the agent in a biological system by reducing or inhibiting degradation and / or reducing or inhibiting clearance of the whole or part(s) of the agent.

[0063] In an embodiment, the agent comprises amino acids. Preferably, the agent is a protein, peptide, bicyclic peptide, tricyclic peptide or a polypeptide. The term "protein" as used herein takes its conventional meaning, namely a plurality of amino acids that are linked together via a peptide bond, to form a polypeptide polymer chain. In an additional embodiment, the agent comprises non-natural isomers or amino acids.

[0064] Polynucleotides which encode suitable therapeutic domains are known in the art or can be readily designed from known sequences such as from sequences of proteins known to interact with surface markers expressed on unwanted cells or contained in nucleotide sequence databases such as the GenBank, EMBL and dbEST databases. Polynucleotides which encode suitable stabilising domains are known in the art or can readily be designed from known sequences and made. Polynucleotides which encode suitable cleavable domains are known in the art or can readily be designed from known sequences and made. Those skilled in the art would be capable of making such agents, which are typically established based on known approaches, such as chemical synthesis techniques and / or genetic engineering techniques.

[0065] In genetic engineering techniques, the nucleic acid is expressed in a suitable host to produce an engineered agent of the invention. Thus, the nucleic acid encoding the agent of the invention may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the agent of the invention of the invention.

[0066] It is appreciated that the nucleic acid encoding the agent of the invention may be joined to a wide variety of other nucleic acid sequences for introduction into an appropriate host. The companion nucleic acid will depend upon the nature of the host, the manner of the introduction of the nucleic acid into the host, and whether episomal maintenance or integration is desired, as is well known in the art. Amino acid residues described herein are generally in the natural "L" isomeric form. However, residues in the "D" isomeric form can be substituted for L-amino acid residues in certain situations, provided that the agent of the invention still retains its function. The definition also includes, unless otherwise specifically indicated, chemically modified amino acids, including amino acid analogues (such as penicillamine, 3-mercapto-D-valine), naturally occurring non- proteogenic amino acids (such as norleucine), beta-amino acids, azapeptides, N-methylated amino acids and chemically synthesised compounds that have properties known in the art to be characteristic of an amino acid. The term "proteogenic" indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways. The definition also includes amino acids in which the functional side group has been chemically derivatised. Such derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulfonyl groups, carbobenzoxy groups, t-butyloxycarbonyl groups, chloroacetyl groups or formyl groups. Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters or hydrazides. Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives. Also included as derivatives are those peptide portions that contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids.

[0067] It is appreciated that the peptide portions of the agent of the invention can be peptide "mimetics", i.e. peptidomimetics which mimic the structural features of peptides comprising or consisting of the amino acid sequence as described herein. Peptidomimetics can be even more advantageous in therapeutic use, in the resistance to degradation, in permeability or in possible oral administration.

[0068] Non protein entities (such as PEG molecules) can be fused using well known methods in the art. Covalent chemical conjugation techniques may include but are not limited to: (i) adding a C-terminal cysteine residue, and conjugating through this via maleimide chemistry, (ii) conjugation through lysines; and / or (iii) using His tags to conjugate.

[0069] As is well known, the glomerular filtration barrier (GFB) is a highly specialised blood filtration interface that displays a high conductance to small and midsized solutes in plasma but retains relative impermeability to macromolecules. Therefore, the barrier enables the renal elimination of small and midsized solutes but stops renal elimination of macromolecules, functioning as a sieve that typically only lets water and small solutes pass through to be cleared by the kidneys. In one embodiment, the agent is a size which prevents it from penetrating the glomerular filtration barrier. Suitable agents above a certain size will be unable to pass through the GFB and therefore will not be eliminated by the kidney. It will be appreciated by those skilled in the art, that the GFB threshold size will vary from one species of animal to the next and may even vary from subject to subject. Moreover, in disease, there can be a change in glomerular permselectivity, thereby making the GFB "leakier" such that macromolecules (e.g. albumin) can be eliminated by the kidney. Other properties of the agent may also have an effect on preventing the agent from penetrating the GFB, for example, the charge, composition, or surface modifications.

[0070] In some embodiments, the agent has a size of at least 6 nanometres (nm), at least 6.5 nm, at least 7 nm, at least 7.5 nm, at least 8 nm, at least 8.5 nm, at least 9 nm, at least 9.5 nm, at least 10 nm. By "size", we include the meaning of the hydrodynamic diameter of the agent. Those skilled in the art would be capable of selecting an appropriate assay to measure the size of the agent. For example, the hydrodynamic diameter may be measured using Dynamic Light Scattering (DLS) or any standard technique in the art.

[0071] In another embodiment, the agent has a molecular weight of at least 40 kilodaltons (kDa), at least 41 kDa, at least 42 kDa, at least 43 kDa, at least 44 kDa, at least 45 kDa, at least 46 kDa, at least 47 kDa, at least 48 kDa, at least 49 kDa, at least 50 kDa, at least 51 kDa, at least 52 kDa, at least 53 kDa, at least 54 kDa, at least 55 kDa, at least 56 kDa, at least 57 kDa, at least 58 kDa, at least 59 kDa, at least 60 kDa, at least 61 kDa, at least 62 kDa, at least 63 kDa, at least 64 kDa, at least 65 kDa, at least 66 kDa, at least 67 kDa, at least 68 kDa, at least 69 kDa, at least 70 kDa, at least 71 kDa, at least 72 kDa, at least 73 kDa, at least 74 kDa, at least 75 kDa, at least 76 kDa, at least 77 kDa, at least 78 kDa, at least 79 kDa, at least 80 kDa, at least 90 kDa, at least 100 kDa, or at least 150 kDa.

[0072] The molecular weight of an agent substance, also called the molar mass, M, is the mass of 1 mole of that substance, given in M gram. The molecular weight of an agent can be measured or calculated by standard techniques known in the art, such as mass spectrometry, methods based on viscosity and light-scattering or sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

[0073] In some embodiments, the half-life of the agent in a biological system is at least 10 hours, at least 12 hours, at least 14 hours, at least 16 hours, at least 18 hours, at least 20 hours, at least 22 hours, at least 24 hours, at least 26 hours, at least 28 hours, at least 30 hours, at least 32 hours, at least 36 hours, at least 38 hours, at least 40 hours, at least 42 hours, at least 44 hours, at least 46 hours, at least 48 hours, at least one week, at least two weeks, at least three weeks, at least four weeks, at least a month, or at least two months.

[0074] In some embodiments, the half-life in a biological system is measured between about 35 °C and about 40 °C, between about 36 °C and about 39 °C, and / or between about 36.5 °C and about 37.5 °C. "Half-life" ("ti / 2", "pharmacokinetic (PK)") is well-known in the art to mean the time taken for the amount of the active agent in the body to decrease by 50%. Those skilled in the art would be capable of selecting an appropriate assay (for example, an enzyme-linked immunosorbent assay (ELISA)) to measure the amount of the agent in the serum at regular intervals over time.

[0075] An agent with a longer half-life will take longer to eliminate from the biological system and thus increase the amount of agent that is exposed to the TME. In some embodiments, the half-life in a biological system of the agent is at least 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or at least 15 times longer, or at least 48, 50, 100, 200, 250, 265, 275, 300 times longer than the half-life of the therapeutic domain when present in a biological system in isolation from (i.e., when not conjugated to) the cleavable domain and / or the stabilisation domain.

[0076] Therapeutic domain

[0077] In some embodiments, the therapeutic domain is a domain that, when present in isolation from the cleavable domain and the stabilisation domain, in a biological system, has a half-life of less than 10 hours, less than 9.5 hours, less than 9 hours, less than 8.5 hours, less than 8 hours, less than 7.5 hours, less than 7 hours, less than 6.5 hours, less than 6 hours, less than 5.5 hours, less than 5 hours, less than 4.5 hours, less than 4 hours, less than 3.5 hours, less than 3 hours, less than 2.5 hours, less than 2 hours, less than 1.5 hours, less than 1 hour, less than 60 minutes, less than 40 minutes, less than 20 minutes, less than 10 minutes, less than 5 minutes, or less than 2 minutes.

[0078] By "penetrate a solid tumour", we include the meaning that the agent or therapeutic domains are taken up by a solid tumour e.g., by diffusion, intracellular transport (e.g. transcytosis), or paracellular transport. Preferably, the agent or therapeutic domains are taken up by a solid tumour by diffusion. Those skilled in the art would be capable of selecting an appropriate method to measure the ability of the agent to penetrate a solid tumour, for example by immunostaining or immunofluorescence of the agent in an in vivo tumour, organoid or tumoroid analysis, a xenograft mouse model or an in an in vitro tumour model.

[0079] By "solid tumour", we include the meaning of heterotypic aggregates of different cell types, including for example, cancer cells, cancer stem cells, connective-tissue cells, and immune cells. In an embodiment, the solid tumour is malignant. In some embodiments, the solid tumour can be a carcinoma, a sarcoma, a lymphoma, or a melanoma.

[0080] In some embodiments, the ability of the therapeutic domain to penetrate a solid tumour when present in isolation from (i.e., when not conjugated to) the cleavable domain and / or the stabilisation domain, is increased relative to the ability of the agent. In some embodiments, the increase is at least 1.5-fold, at least 2-fold, at least 2.5-fold, at least 2-fold, at least 2.5-fold, at least 3-fold, at least 3.5-fold, at least 4-fold, at least 4.5-fold, at least a 5-fold increase, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least a 10-fold increase, at least a 15-fold increase, at least a 20-fold increase, at least 25-fold, at least 30-fold, or at least 35-fold.

[0081] 'Tumour uptake level' is the amount of administered therapeutic agent that is actually taken up by the tumour. In some embodiments, the tumour uptake level of the therapeutic domain when cleaved from the agent is at least 8%ID / g, preferably at least 9%ID / g, more preferably at 10%ID / g. By "%ID / g", we include the meaning of average concentration of total antibody (bound + free) in the tumour (Schmidt and Wittrup, 2009). Those skilled in the art would be capable of selecting an appropriate method to measure the tumour uptake level, for example by immunostaining, immunofluorescence or radioactive labelling of the agent in an in vivo tumour, a xenograft mouse model or an in an in vitro tumour model.

[0082] In some embodiments, the increased penetrability and / or tumour uptake levels are sustained. In another embodiment, the increased penetrability and / or tumour uptake levels are transient. By "sustained", we include the meaning that the increased levels of the agent are maintained over time and / or continue to penetrate the solid tumour. By "maintained over time", we include the meaning of the increased levels being maintained for a time of at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least 11 hours, at least 12 hours, at least 24 hours, at least 48 hours, or at least 36 hours. By "transient", we include the meaning that the increased levels of the agent are not maintained and / or decrease over time, for example after 20 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 1 minute or less, or 0.5 minutes or less.

[0083] In some embodiments, after cleavage of the therapeutic domain from the agent, the tumour uptake level of the therapeutic domain is increased relative to the tumour uptake level of the agent. In some embodiments, the increase is at least at least 5-fold, at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, at least 10-fold, at least 15-fold, at least a 20-fold, at least 25-fold, at least 30-fold, or at least 35-fold.

[0084] Preferably, the therapeutic domain is selected from any one or more of: i. an antigen-binding domain; ii. a Fab region; iii. a F(ab')2 region; iv. a scFv region; v. a tandem scFv region; vi. a domain antibody, preferably a single domain antibody (sdAb); vii. a nanobody; viii. a monoclonal antibody; ix. a polyclonal antibody; x. a diabody; xi. a triabody; xii. A tetrabody; xiii. A pentabody; xiv. A hexabody; xv. an antibody drug conjugate; xvi. a bispecific peptide, such as a bispecific antibody; xvii. a multispecific peptide, such as a multispecific antibody; xx. a bicyclic peptide; xxi. a tricyclic peptide. Most preferably, the therapeutic domain is any one of i. an antigen-binding domain; ii. a Fab region; iii. a F(ab')2 region; iv. a scFv region; v. a tandem scFv region. In one embodiment, the therapeutic domain is not a single domain antibody (sdAb).

[0085] In a preferred embodiment, the therapeutic domain is (or is derived from) a Fab, a ScFv, a VHH, a T-cell receptor (TCR), an antibody mimic, or any domain thought to bind an antigen in order to either agonise or antagonise a receptor signal pathway to treat disease. In any embodiment herein, the therapeutic domain comprises means for binding an antigen, for example any of the antigens described elsewhere herein.

[0086] Preferably, the therapeutic domain is or is derived from a Fab region, a F(ab')2 region or a scFv region.

[0087] In one embodiment, the therapeutic domain comprises no more than one binding domain. In a further embodiment, the therapeutic domain does act as a cross-linker between the tumour and T cells.

[0088] In some embodiments, the therapeutic region further comprises a hinge region. The term "hinge region" includes the stretch of amino acids in the heavy chain of an antibody between the antigen-binding portion (e.g. Fab) and the Fc domain.

[0089] By "antigen-binding domains", we include the meaning of domains that can bind to an antigen. Such domains include antibody parts and non-antibody parts. By "antibody parts thereof", we include the meaning of an antibody fragments (such as Fab, Fv, ScFv, dAb, nanobodies). By "non-antibody parts", we include the meaning of non-antibody type scaffolds such as affibodies, affilins, anticalins, atrimers, DARPins, FN3 scaffolds (such as adnectins and centyrins), fynomers, kunitz domains, pronectins, obodies, inhibitor cystine knots (also known as cys-knots or knottins), thyrodoxin repeats, fibronectin domains, lipocalin.

[0090] Fragment antigen-binding (Fab) regions are regions of an antibody that bind to antigens. A Fab region is composed of one constant domain and one variable domain of each of the heavy chain and the light chain (Figure 1). Fab regions have a monovalent epitope binding site.

[0091] Preferably, the Fab comprises or consists of a heavy chain and a light chain, wherein the heavy chain and light chain are covalently connected via a C-terminal cysteine residues at position 5 (IMGT hinge numbering), position 220 (EU numbering), position 233 (Kabat numbering) and / or position 233 (Chothia numbering) of the heavy chain. The C-terminal cysteine of the heavy chain forms an inter-chain linkage with a cysteine residue on the light chain. The light chain may be a kappa light chain or a lambda light chain. In an embodiment, the light chain is a kappa light chain and the cysteine residue that forms the inter-chain linkage is at position 126 (IMGT unique numbering for C domain for kappa light chains), 215 (EU numbering), 214 (Kabat numbering) and / or 214 (Chothia numbering). In an embodiment, the light chain is a lambda light chain and the cysteine residue that forms the inter-chain linkage is at position 126 (IMGT unique numbering for C-domain of lambda light chains), 214 (EU numbering), 214 (Kabat numbering) and / or 214 (Chothia numbering). In a preferred embodiment, there are no further C or N-terminal (in particular N-terminal) appendages to the Fab domain (i.e. other than the cleavage domain and / or the cleavage domain and stabilisation domain). In another embodiment, there is no more than 4 (e.g. no more than 3, or no more than 2) N-terminal amino acid residues to the Fab.

[0092] Divalent antibody (F(ab')2) regions are Fab regions with additional amino acids which are linked to each other (Figure 2). F(ab')2 regions may include the entire hinge region that holds the two heavy chains together.

[0093] A single-chain variable (scFv) region is a fusion protein of the variable regions of the heavy chain (VH) and light chain (VL) of an antibody. In an embodiment, the heavy chain comprises or consists of residues in positions 0 to 113 (Kabat numbering), positions 0 to 113 (Chothia numbering), positions 1 to 117 (EU numbering) and / or position 1 (IMGT unique numbering for the V-domain) to position 1.5 (IMGT unique numbering for the C-domain). In an embodiment, the light chain comprises or consists of residues in positions 1 to 109 (Kabat numbering), positions 1 to 109 (Chothia numbering), positions 1 to 109 (EU numbering) and / or position 1 (IMGT light chain V-domain numbering) to position 1.3 (IMGT C-domain numbering).

[0094] In an scFv, the two chains are connected with a short linker peptide. Preferably the heavy chain variable domain and the light chain variable domain are covalently attached through a flexible neutral linker. scFv regions can be in the VL-VH or VH-VL orientation. In an embodiment, the flexible linker allows for the self-assembly of a heavy and light chain variable region. In an embodiment, the linker length is a minimal distance of 3.5 nm and / or is at least 12 amino acids. In an embodiment the linker has a maximum length of 30 amino acids. In an embodiment, the linker is 12-30 amino acids long. In another embodiment the linker is 12-25 amino acids long. In a preferred embodiment, the linker is 15-20 amino acids long. In an embodiment, the linker is located after position 113 (Kabat numbering), position 113 (Chothia numbering), position 117 (EU numbering) and / or position 1.5 (IMGT unique numbering for the C-domain), if the heavy chain is the N-terminal domain. In an alternative embodiment, the linker is located after position 109 (Kabat numbering), position 109 (Chothia numbering), position 109 (EU numbering) and / or position 1.3 (IMGT C-domain numbering) if the N-terminal domain is a light chain, either kappa or lambda. scFv regions can be monovalent (scFv), or multivalent such as bivalent (e.g. tandem scFv, di- scFv or bi-scFv, diabody), trivalent (e.g. triabody, tribody or tri-scFv), or tetravalent (e.g. tetrabody), pentavalent (e.g. pentabody), hexavalent (e.g. hexabody).

[0095] The scFvs may be expressed as single domains comprising a N and / or C-terminal appendage. The appendage may be a purification and / or detection tag, such as HIS tags, GST tags, myc tags etc. The purification tag or detection tag may be any tag suitable tag known in the art, e.g. that aids in purification and / or detection. In one embodiment the scFvs are expressed as domains without a N and / or C-terminal appendage, in particular without any purification and / or detection tag.

[0096] Antibody drug conjugates (ADCs) are molecules comprising an antibody linked to a biologically active cytotoxic drug. For use in preventing or treating conditions characterised by the presence of unwanted cells (e.g. cancer), the antibody is specific to antigens expressed on the unwanted cells and guides the cytotoxic drug to the required location in the body. Examples of ADCs may include but are not limited to trastuzumab emtansine (also called Kadcyla - made by Genentech or Roche), enfortumab vedotin (also called Padcev - made by Astellas or Seattle Genetics), trastuzumab deruxtecan (also called Enhertu - made by AstraZeneca / Daiichi Sankyo), Sacituzumab govitecan (also called Trodelvy - made by Immunomedics), belantamab mafodotin (also called Blenrep - made by GlaxoSmithKline), Tisotumab vedotin- tftv (also called Tivdak - made by Seagen Inc).

[0097] A single-domain antibody (sdAb), also known as a nanobody, is an antibody fragment consisting of a single monomeric variable antibody domain. They can be derived from VH domains (e.g. VHH (or VHH) fragments from camelids such as llamas, VNAR fragments from cartilaginous fish), VL domains, or human domain antibodies.

[0098] In an embodiment, the therapeutic domain is a domain antibody, nanobody or VHH, which comprises or consists of either a heavy chain or a light chain, and three CDR sequences. The light chain may be a kappa light chain or a lambda chain.

[0099] In an embodiment, the heavy chain comprises or consists of residues at positions 0 to 113 (Kabat numbering), positions 0 to 113 (Chothia numbering), positions 1 to 117 (EU numbering) and / or positions 1 (IMGT unique numbering for the V-domain) to 1.5 (IMGT unique numbering for the C-domain). In one embodiment, the heavy chain is 85, 90 or 95% of the length of the chain described above.

[0100] In an embodiment, the light chain comprises or consists of residues at position 1 to 109 (Kabat numbering), positions 1 to 109 (Chothia numbering), positions 1 to 109 (EU numbering) and / or position 1 (IMGT light chain V-domain numbering) to 1.3 (IMGT C-domain numbering). In one embodiment, the light chain is 85, 90 or 95% of the length of the chain described above.

[0101] Preferably, the heavy chain comprises three complementarity determining regions (CDRs):

[0102] • a HCDR1 at positions H27-38 (IMGT), positions H31-H35b (Kabat), and / or positions H26-H32 (Chothia);

[0103] • a HCR2 at positions H56-65 (IMGT), H50-H65 (Kabat), and / or positions H52-5 (Chothia); and

[0104] • a HCDR3 at positions 105-115 (IMGT), positions H95-102 (Kabat), and / or positions H96-101, for CDRH3.

[0105] Preferably, the light chain comprises three complementarity determining regions (CDRs):

[0106] • a LCDR1 at positions L27-38 (IMGT), positions L24-34 (Kabat), and / or positions L26- L32 (Chothia);

[0107] • a LCDR2 at positions L56-65 (IMGT), positions L50-56 (Kabat) and / or positions L50- 52 (Chothia); and

[0108] • a LCDR3 at positions 105-111 (IMGT), positions L89-97 (Kabat), and / or positions 91-96 (Chothia).

[0109] In an embodiment, the VHH, nanobody or domain antibody contains a mutation that increases the stability and / or enhances monomeric expression and / or enhances the therapeutic potential. The skilled person would be able to select suitable mutations from the prior art. For example, the mutations may be in residues that are located on the interface between the variable heavy and light chains. The mutation may involve mutating these residues to a (more) soluble amino acid or residues on equivalent positions on naturally occurring VHH antibodies from camelids, llamas, shark, or other related organisms that contain a VHH domain naturally.

[0110] Nanobodies, VHHs or domain antibodies, if expressed as single domains, may comprise a N and / or C-terminal appendage. The appendage may be a purification and / or detection tag, such as HIS tags, GST tags, myc tags etc. The purification tag or detection tag may be any suitable tag known in the art, e.g. that aids in purification and / or detection. In an embodiment, the therapeutic domain does not comprise a purification tag or detection tag, (for example, the therapeutic domain is a VHH, nanobody or domain antibody in which a purification and / or detection tag has been removed). The skilled person would be aware of suitable methods for removing purification tags or detection tags (e.g. enzymatic cleavage).

[0111] A monoclonal antibody (mAb) is an antibody produced from a cell lineage made by cloning a unique cell, or through a humanised mouse, or recombinant in vitro technology (such as phage display, yeast display or mammalian display), or from in vitro displays (such as ribosome display, mRNA display, cis display). Monoclonal antibodies can have monovalent affinity, binding only to the same epitope (monospecific). In contrast, polyclonal antibodies bind to multiple epitopes (multispecific) and are usually made by several different antibody-secreting plasma cell lineages. In some embodiments, the monoclonal antibodies can be engineered to be multispecific in order to increase the number of epitopes that it binds to.

[0112] The therapeutic domain may be monospecific or multispecific, for example bispecific or trispecific. By "multispecific" we include the meaning that the peptide (such as an antibody or antibody fragment) is specific for two or more antigens (for example, bispecific tandem di- scFvs). In one embodiment, the therapeutic domain is not multispecific.

[0113] Bicyclic and tricyclic peptides are synthetic short peptides constrained to form two or three loops respectively using a chemical connector compound known as a scaffold, which stabilises their structural geometry. Examples of bicyclic peptides include but are not limited to BT8009 (anti-Nectin 4), BT1718 (anti-MTl-MMP) and BT5528 (anti-EphA2).

[0114] In some embodiments, the therapeutic domain has a molecular weight of less than or equal to 1 kDa, less than or equal to 2 kDa, less than or equal to 3 kDa, less than or equal to 4 kDa, less than or equal to 5 kDa, less than or equal to 6 kDa, less than or equal to 7 kDa, less than or equal to 8 kDa, less than or equal to 9 kDa, less than or equal to 10 kDa, less than or equal to 15 kDa, less than or equal to 20 kDa, less than or equal to 25 kDa, less than or equal to 27 kDa, less than or equal to 30 kDa, less than or equal to 35 kDa, less than or equal to 40 kDa, less than or equal to 45 kDa, less than or equal to 50 kDa, less than or equal to 55 kDa, less than or equal to 60 kDa, less than or equal to 65 kDa, less than or equal to 70 kDa, less than or equal to 75 kDa, less than or equal to 80 kDa, less than or equal to 85 kDa, less than or equal to 90 kDa, less than or equal to 95 kDa, or less than or equal to 100 kDa.

[0115] In one embodiment, the cleaved therapeutic domain has a molecular weight of no more than 60 kDa, no more than 55 kDa, no more than 50 kDa, no more than 40 kDa, no more than 30 kDa, no more than 25 kDa, no more than 20 kDa, no more than 20 kDa, or no more than 15 kDa.

[0116] Preferably, the agent is selected from the group: i. a monoclonal antibody; ii. a polyclonal antibody; iii. diabody; iv. triabody; v. tetrabody; vi. pentabody; vii. hexabody; viii. an antibody drug conjugate; ix. a bispecific peptide, such as a bispecific antibody; and / or x. a multispecific peptide, such as a multispecific antibody.

[0117] Particularly preferred agents and / or therapeutic domains include antibodies that are derived from anti-epidermal growth factor receptor (EGFR) antibodies such as Cetuximab-derived fragments, Panitumumab-derived fragments, Zalutumumab-derived fragments; from anti- HER.2 antibodies such as Trastuzumab-derived fragments, Pertuzumab-derived fragments; from anti-CD20 antibodies such as Rituximab-derived fragments; from anti-CD22 antibodies such as Inotuzumab-derived fragments; from anti-CD70 antibodies; from anti-CD33 antibodies such as hp67.6-derived fragments, Gemtuzumab-derived fragment; from anti-MUCl antibodies such as GP1.4- derived fragments, SM3- derived fragments; from anti-CD40 antibodies, from anti-CD74 antibodies, from anti-P-cadherin antibodies, from anti-EpCAM antibodies; from anti- CD138 antibodies; from anti-E-cadherin antibodies; from anti-CEA antibodies such as M5A; fromanti-FGFR3 antibodies; from anti-PSMA antibodies such as J591; from anti-CTLA4 antibodies, such as Ipilimumab-derived fragments, Tremelimumab-derived fragments; from anti-PDl antibodies, such as Pembrolizumab-derived fragments, Nivolumab-derived fragments; and from anti-PDLl antibodies, such as Atezolizumab-derived fragments; from anti- VEGF (vascular endothelial growth factor) antibodies, such as Bevacizumab.

[0118] By "antibodies that are derived from" and "antibody-derived fragment", we include the meaning of antibodies or fragments of antibodies that comprise parts or modified parts of the parent antibody from which they are derived. For example, an antibody-derived fragment may have the same of similar sequences as parts of the parent antibody. In some embodiments, an antibody-derived fragment has at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% or 79% sequence identity to a part of the antibody sequence. In other embodiments, an antibody-derived fragment has at least 80%, 82%, 85%, 87% or 89% sequence identity to a part of the parental antibody sequence. In other embodiments, an antibody-derived fragment has at least 90%, 92%, 95%, 97% or 99% sequence identity to a part of the parental antibody sequence. In a particular embodiment, an antibody derived fragment is identical to the corresponding part of the parental antibody.

[0119] In some embodiments, the agents provided herein in an uncleaved state comprise a Trastuzumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from the group consisting of:

[0120] The cleavable domains and the cleavage sites of the below described engineered designs Dl- D9, Dlx-D7x and D12-D20 have been emboldened and underlined, respectively.

[0121] Dlx: Engineered trastuzumab antibody design lx (SEQ ID NO: 1)

[0122] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSLSGRSDNHDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIE KTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Trastuzumab Design 1 (DI) Protein sequence - HC002 - (SEQ ID NO: 105)

[0123] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0124] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0125] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSLSGRSDNHASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV

[0126] VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0127] D2x: Engineered trastuzumab antibody design 2x (SEQ ID NO: 2)

[0128] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0129] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0130] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCLSGRSDNHDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0131] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0132] >Trastuzumab Design 2 (D2) protein sequence - AA004 - (SEQ ID NO: 107)

[0133] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0134] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0135] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCLSGRSDNHASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD

[0136] VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0137] D3x: Engineered trastuzumab antibody design 3x (SEQ ID NO: 3)

[0138] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0139] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0140] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSLSGRSDNHGSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV

[0141] VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0142] >Trastuzumab Design 3 (D3) protein sequence - HC004 - (SEQ ID NO: 109)

[0143] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0144] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

[0145] SNTKVDKKVEPKSCGSLSGRSDNHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT

[0146] CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP

[0147] APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0148] D4x: Engineered trastuzumab antibody design 4x (SEQ ID NO: 4)

[0149] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0150] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0151] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

[0152] SNTKVDKKVEPKSCLSGRSDNHCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

[0153] VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0154] >Trastuzumab Design 4 (D4) protein sequence - HC005 - (SEQ ID NO: 111)

[0155] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0156] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0157] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

[0158] SNTKVDKRVEPKSCLSGRSDNHCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE

[0159] VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0160] D5x: Engineered trastuzumab antibody design 5x (SEQ ID NO: 5)

[0161] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0162] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0163] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

[0164] SNTKVDKKVEPKSCLSGRSDNHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP

[0165] EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG

[0166] QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0167] >Trastuzumab Design 5 (D5) protein sequence - HC007 - (SEQ ID NO: 113)

[0168] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0169] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0170] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCLSGRSDNHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0171] D6x: Engineered trastuzumab antibody design 6x (SEQ ID NO: 6)

[0172] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPLSGRSDNHELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0173] In the engineered trastuzumab antibody design 6x (D6x), LSGRSDNH (SEQ ID NO: 77) was added to the CH2 domain three amino acids (PAP) after the hinge disulphide bond region (CPPC (SEQ ID NO: 79)) (to give a F(ab')2 fragment) to allow accessibility of the protease. The protease cleavage site as added after residue 1.5 of the IMGT unique numbering for the C- domain, residue 232 according to the EU numbering scheme and 245 according to the Kabat and Chothia numbering schemes. The protease cleavage site was also added before residue 1.4 from the IMGT unique numbering for the C-domain, residue 232 according to the EU numbering scheme and 246 according to the Kabat and Chothia numbering schemes.

[0174] >Trastuzumab Design 6 (D6) protein sequence - HC021 - (SEQ ID NO: 115)

[0175] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPALSGRSDNHPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0176] D7x: Engineered trastuzumab antibody design 7x (SEQ ID NO: 7)

[0177] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAPLSGRSDNHFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGN VFSCSVM H EALH N HYTQKSLSLSPG K In the engineered trastuzumab antibody design 7x (D7x), ELLGGPSV (SEQ ID NO: 81) was substituted with LSGRSDNH (SEQ ID NO: 77) three amino acids (PAP) after the hinge disulphide bond region (CPPC (SEQ ID NO: 79)). This is the minimal mutation that could be made to give a F(ab')2 fragment. Residues 1.5-4 from the IMGT unique numbering for the C- domain were substituted with a protease sequence, or residues 233-240 from the EU numbering scheme, or residues 246-253 from the Kabat and Chothia numbering schemes.

[0178] >Trastuzumab Design 7 (D7) protein sequence - HC020 - (SEQ ID NO: 117)

[0179] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPALSGRSDNHFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGN VFSCSVM H EALH N HYTQKSLSLSPG K

[0180] D9: Engineered trastuzumab antibody design 9 [ADAM10] (SEQ ID NO: 8)

[0181] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSPRAEALKGGGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0182] D17: Engineered anti-EGFR antibody design 17 [VHH 7D12-FC] (SEQ ID NO: 9)

[0183] QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRF TISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSSLSGRSD NHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

[0184] D18: Engineered trastuzumab antibody design 18 [anti-HER2 ScFv-FC] (SEQ ID NO: 10) DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLR AEDTAVYYCSRWGGDGFYAMDYWGOGTLVTVSSGGGSGGGSLSGRSDNHGSASDKTHTCPPCPA PELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR VVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK

[0185] In some embodiments, the agents provided herein in an uncleaved state comprise an trastuzumab-derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequences above (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 105, 107, 109, 111, 113, 115, and / or 117), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0186] In some embodiments, the agents provided herein in an uncleaved state comprise a Cetuximab- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0187] D12: Engineered cetuximab antibody design 12 (SEQ ID NO: 11):

[0188] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSCGSLSGRSDNHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTC VVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPA PIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0189] In some embodiments, the agents provided herein in an uncleaved state comprise a cetuximab- derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequence above (SEQ ID NO: 11), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0190] In some embodiments, the agents provided herein in an uncleaved state comprise a Bevacizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0191] D13: Engineered bevacizumab antibody design 13 (SEQ ID NO: 12) :

[0192] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFT FSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSCGSLSGRSDNHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL

[0193] DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0194] In some embodiments, the agents provided herein in an uncleaved state comprise a Bevacizumab-derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequence above (SEQ ID NO: 12), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0195] In some embodiments, the agents provided herein in an uncleaved state comprise a Pembrolizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from the group consisting of:

[0196] D14a : Engineered pembrolizumab antibody design 14a (SEQ ID NO: 13) :

[0197] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGSLSGRSDNHGSGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0198] D14b: Engineered pembrolizumab antibody design 14b (SEQ ID NO: 14) :

[0199] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKYGPPGSLSGRSDNHGSCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEK TISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0200] In some embodiments, the agents provided herein in an uncleaved state comprise a pembrolizumab-derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequences above (SEQ ID NOs: 13 and 14), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0201] In some embodiments, the agents provided herein in an uncleaved state comprise a Pembrolizumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence: D14c: Engineered Pembrolizumab antibody design 14C (SEQ ID NO: 97) :

[0202] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCGSLSG RSDNHGSNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDV SQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKT ISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFL YSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0203] In some embodiments, the agents provided herein in an uncleaved state comprise a Pembrolizumab-derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequence above (SEQ ID NO: 97), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0204] In some embodiments, the agents provided herein in an uncleaved state comprise a MSA- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0205] D15: Engineered M5A antibody design 15 (SEQ ID NO: 15) :

[0206] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSCGSLSGRSDNHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVL DSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0207] In some embodiments, the agents provided herein in an uncleaved state comprise an MSA- derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequence above (SEQ ID NO: 15), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0208] In some embodiments, the agents provided herein in an uncleaved state comprise an Ipilimumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0209] D16: Engineered Ipilimumab antibody design 16 (SEQ ID NO: 16): QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSCGSLSGRSDNHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPI EKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0210] In some embodiments, the agents provided herein in an uncleaved state comprise a Ipilimumab-derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequence above (SEQ ID NO: 16), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0211] In some embodiments, the agents provided herein in an uncleaved state comprise an anti- EGFR-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0212] D17: Engineered anti-EGFR antibody design 17 [VHH 7D12-FC] (SEQ ID NO: 9)

[0213] QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRF TISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTOVTVSSSLSGRSD NHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPP SREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK

[0214] In some embodiments, the agents provided herein in an uncleaved state comprise an anti- EGFR-derived variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequence above (SEQ ID NO: 9), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0215] In some embodiments, the agents provided herein in an uncleaved state comprise a trastuzumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0216] D20: Engineered PEGylated trastuzumab scFv design 20 (SEQ ID NO: 98) :

[0217] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSD IQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGSGGGSLSGRSDNHGGGSHHHHHHHH

[0218] In some embodiments, the agents provided herein in an uncleaved state comprise an variant that comprises up to and including 50 amino acid substitutions, insertions or deletions relative to the sequences above (i.e. any of the sequences selected from SEQ ID NOS: 1 to 16, 97 and 98), for example up to and including : 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 (in particular 1 to 5) amino acid substitutions, insertions and / or deletions.

[0219] In some embodiments, the amino acid insertions and / or deletions of the variant outlined above are located in the therapeutic domain of the variant optionally in such an embodiment, the cleavable domain and / or the stabilisation domain may be unchanged relative to any of the parental sequences of SEQ ID NOs: 1-16, 97 and 98.

[0220] In some embodiments, the amino acid insertions and / or deletions of the variant outlined above are located in the cleavable domain of the variant, optionally in such an embodiment, the therapeutic domain and / or the stabilisation domain may be unchanged relative to any of the parental sequences of SEQ ID NOs: 1-16, 97 and 98.

[0221] In some embodiments, the amino acid insertions and / or deletions of the variant outlined above are located in the stabilisation domain of the variant, optionally in such an embodiment, the cleavable domain and / or the therapeutic domain may be unchanged relative to any of the parental sequences of SEQ ID NOs: 1-16, 97 and 98.

[0222] In some embodiments the linker domains of the variant may be unchanged relative to any of the parental sequences of SEQ ID NOs: 1-16, 97 and 98. In other embodiments the amino acid insertions and / or deletions of the variant outlined above are located in the linker domains of any of the of sequences of SEQ ID NOs: 1-16, 97 and 98.

[0223] In some embodiments, the therapeutic domain is monovalent, bivalent, trivalent or multivalent (e.g. tetravalent). A monovalent therapeutic domain may have an affinity for one epitope, antigen, or strain of microorganism; whereas, a multivalent (e.g. bivalent, trivalent, tetravalent) therapeutic domain may have an affinity for various epitopes, antigens, or strains of microorganisms. In one embodiment, the therapeutic domain is not multivalent.

[0224] In one embodiment, the therapeutic domain targets or guides the agent to unwanted cells. By "therapeutic domain targeting the agent", we include the meaning that that therapeutic domain may by a specific binding partner of an entity (e.g. target) expressed by or associated with unwanted cells. In one embodiment, the therapeutic domain is a specific binding partner of an entity (e.g. target) expressed by or associated with a target cell or a target tissue. Typically, the expressed entity is expressed selectively on the unwanted cell. For example, the abundance of the expressed entity (e.g. target and / or antigen) is typically 10 or 100 or 500 or 1,000 or 5,000 or 10,000-fold higher on the unwanted cell than on other cells within the body to be treated. However, as mentioned below, the cleavage site provides additional specificity on where the therapeutic domain is released and so the binding partner may bind an entity (e.g. target and / or antigen) that is similarly or under-expressed on unwanted cells relative to other cells within the body.

[0225] Most preferably, however, the therapeutic domain is a specific binding partner of an entity (e.g. target and / or antigen) expressed by or associated with unwanted cells, as opposed to any other cells.

[0226] By "binding partner" we include the meaning of a molecule that binds to a target entity (e.g. antigen) expressed by a particular cell. Preferably, the binding partner binds selectively to that entity. Antibodies that bind specifically to a target (which can be an epitope) are antibodies which bind to that target with greater affinity, avidity, more readily, and / or with greater duration than to other unrelated targets or molecules.

[0227] As will be appreciated, the specificity of an antibody for its target can be determined and / or defined based on affinity measurements. Affinity (KD), expressed by the equilibrium constant for dissociation between antigen and antibody, is a measure of the strength of binding between the epitope and the antigen binding site on the antibody: a smaller KD value indicates that the binding strength between antigen binding molecules is stronger (alternatively, affinity can also be expressed as an affinity constant (KA), which is 1 / KD) . AS will be apparent to those skilled in the art, affinity can be determined by any method known in the art and described herein. Any KD value greater than IxlO'6M is generally considered to indicate non-specific binding.

[0228] For example, it is preferred if the binding partner has a KD value in respect of the target which is at least five or ten times lower (i.e. higher affinity) than for at least one other entity expressed by another cell (e.g. a normal cell type), and preferably more than 100 or 500 times lower. More preferably, the binding partner of that entity has a KD value more than 1000 or 5000 times lower than for at least one other entity expressed by another cell (e.g. normal cell type). Binding specificity of the binding molecule can be determined experimentally by methods known in the art. Such methods comprise but are not limited to Biophysical Biolayer interferometry (BLI), isothermal titration calorimetry (ITC), Western blots, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), electrochemiluminescence (ECL), immunoradiometric assay (IRMA), Enzyme immunoassay (EIA), and surface plasmon resonance (SPR). Typically, the binding partner is one that binds to an entity that is present or accessible to the binding partner in significantly greater concentrations in or on or around unwanted cells than in any normal cells of the host. For example, the binding partner may bind to a tumour associated antigen which is expressed on the cell membrane. In another embodiment, the binding partner may bind an entity that is similarly or under-expressed on unwanted cells relative to other cells within the body. In one embodiment, the entity is naturally occurring or endogenously expressed.

[0229] Preferably, the target entity is an antigen at the unwanted cell surface, i.e. a cell surface marker. The diameter of solid tumours can vary from small (such as 1 centimetre (cm)) to large (such as bigger than 10 cm). Solid tumours are 3D structures, with periphery and interior parts. Target entities may be located at the periphery (e.g. surface) and / or at the interior (e.g. epicentre) of the tumour. The epicentre is considered the inner part of the interior of tumour and may be at least 0.5 cm, at least 1 cm, at least 2 cm, at least 3 cm, at least 4 cm, at least 5 cm, at least 6 cm, or at least 7 cm away from the surface of the tumour (Sopik and Narod, 2018, The relationship between tumour size, nodal status and distant metastases: on the origins of breast cancer, Breast Cancer Res Treat, 2018; 170(3): 647-656).

[0230] "Unwanted cells" include cells whose presence in a host or patient is undesired, such as tumour cells or other disease-causing cells.

[0231] In some embodiments, the therapeutic domain may be any compound or part that specifically binds (in a non-immune sense) to an entity (e.g. target and / or antigen) expressed by unwanted cells or otherwise becomes associated with unwanted cells. Thus, the therapeutic domain may be any of: i. a T-cell receptor (TCR) domain; or ii. a receptor domain; or iii. a receptor mimic domain; or iv. a cytokine; or v. a hormone; or vi. a growth factor; or vii. a peptide; or viii. a derivative of a peptide.

[0232] In one embodiment, the therapeutic domain may bind an intracellular target. In another embodiment, the therapeutic domain may bind an extracellular target. In some embodiments, the therapeutic domain may bind both intracellular and extracellular targets.

[0233] In another embodiment, the therapeutic domain does not target the agent to the unwanted cells.

[0234] Particularly useful therapeutic domains could include peptides or derivatives of peptides such as MYC (also known as c-Myc) inhibitors (e.g. Hl peptide, OmoMYC), HOX (homeobox) inhibitors (e.g. HRX9, HTL-001 (HOX Therapeutics Ltd.)). Particularly useful therapeutic domain targets could include cytokines such as IGF (insulin-like growth factor), EGF (epidermal growth factor), VEGF (vascular endothelial growth factor), IL (interleukin)-2, IL-6, IL-4, or HGF (hepatocyte growth factor, scatter factor, SF, hepatopoeitin A).

[0235] Insulin like growth factors (IGF-1 and IGF-11) are preferentially taken up by malignant cells and so may be used to target tumour cells. Similarly, EGF can be used to target malignant cells which upregulate the EGF receptor. Also, tumour associated blood vessels overexpress VEGF receptor and so can be targeted by the family of VEGF growth factors. Myeloma cells express IL-6 receptor and also secrete IL-6 which acts in an autocrine fashion to stimulate cell proliferation. Thus IL-6 may be used as a therapeutic domain for myeloma. In another example, the therapeutic domain is melanoma stimulating hormone (MSH) which binds to the MSH receptor which is expressed in high numbers in melanoma cells.

[0236] In an embodiment, the therapeutic domain binds to an antigen expressed by the unwanted cell selected from a list comprising: CEA (anticarcinoembryonic antigen); HER2 / Neu; CD22 (sialic acid binding Ig-like lectin 2, SIGLEC2, SIGLEC-2, B-lymphocyte cell adhesion molecule, BL- CAM, Leu-14); EPCAM (epithelial cell adhesion molecule, tumour-associated calcium signal transducer 1, TACSTD1, gastrointestinal tumour-associated protein 2, GA733-2, epithelial glycoprotein 2, EGP-2, epithelial cell adhesion molecule, Ep-CAM, KSA, KS1 / 4 antigen, M4S, tumour antigen 17-1A, EpCAM, CD326); EGFR (epidermal growth factor receptor, receptor tyrosine-protein kinase erbB-1, ERBB1, HER1, HER-1, ERBB); PMSA; CTLA-4 (cytotoxic T lymphocyte-associated antigen 4, CTLA4, CD152) CD30; CD20; CD33 (sialic acid binding Ig- like lectin 3, SIGLEC3, SIGLEC-3, gpG7, p67); CD80 (B7-1, CD28LG1); CD86 (B7-2, CD28LG2); CD2; CA125; Carbonic Anhydrase IX; CD70 (tumour necrosis factor superfamily member 7, TNFSF7, CD27LG, CD27L); CD74 (major histocompatibility class II invariant chain, MH2); CD56; CD40 (tumour necrosis factor receptor superfamily member 5, TNFRSF5, p50); CD19; c-met / HGFR; TRAIL-R1; DR5; PD-1; PD1L; IGF-1R; VEGF; VEGF-R2; Prostate stem cell antigen (PSCA); MUC1 sialylated carbohydrate, tumour-associated (CA242, cancer antigen 242); CanAg; Mesothelin; P-cadherin; Myostatin (GDF8); Cripto (TDGF1); ACVRL1 / ALK1; MUC5AC; CEACAM ((carcinoembryonic antigen-related cell adhesion molecules); CD137; CXCR4; Neuropilin; Glypicans; HER3 / EGFR; PDGFRa (platelet-derived growth factor receptor alpha subunit, PDGFR2, CD140a); EphA2; CD138. Preferably, therapeutic domain binds to an antigen selected from a list comprising HER2, VEGF, PD-1, PMSA, CEA, CTLA-4 or EGFR.

[0237] In some embodiments, the therapeutic domain provided herein comprise a Trastuzumab- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to a sequence selected from the group consisting of: Therapeutic domain of Engineered trastuzumab designs 1-5, lx-5x and 8-10 (SEQ ID

[0238] NO: 17)

[0239] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSC

[0240] Therapeutic domain of Engineered trastuzumab designs 6x & 7x (SEQ ID NO: 18)

[0241] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCDKTHTCPPCPAP

[0242] Therapeutic domain of Engineered trastuzumab designs 6 & 7 (SEQ ID NO: 145)

[0243] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPAP

[0244] Therapeutic domain of Engineered trastuzumab design 17 (SEQ ID NO: 19)

[0245] QVQLQESGGGLVQPGGSLRLSCAASGRTFSSYAMGWFRQAPGKQREFVAAIRWSGGYTYYTDSVKGRF TISRDNAKTTVYLQMNSLKPEDTAVYYCAATYLSSDYSRYALPQRPLDYDYWGQGTQVTVSSS

[0246] Therapeutic domain of Engineered trastuzumab design 18 (SEQ ID NO: 20)

[0247] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKGGGGSGGGGSGGGGSEVQLVESGGGLVQPGGS LRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLR AEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0248] In some embodiments, the therapeutic domain provided herein comprise a Cetuximab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0249] Therapeutic domain of Engineered cetuximab design 12 (SEQ ID NO: 21)

[0250] QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSI NKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSG GTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPS NTKVDKKVEPKSC In some embodiments, the therapeutic domain provided herein comprise a Bevacizumab- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0251] Therapeutic domain of Engineered bevacizumab design 13 (SEQ ID NO: 22)

[0252] EVQLVESGGGLVQPGGSLRLSCAASGYTFTNYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFT FSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPHYYGSSHWYFDVWGQGTLVTVSSASTKGPSVFPLAPSSK STSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVN HKPSNTKVDKKVEPKSC

[0253] In some embodiments, the therapeutic domain provided herein comprise a Pembrolizumab- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to sequence:

[0254] Therapeutic domain of Engineered pembrolizumab designs 14a andl4b (SEQ ID NO: 23)

[0255] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKP SNTKVDKRVESKY

[0256] In some embodiments, the therapeutic domain provided herein comprise a Pembrolizumab- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to sequence:

[0257] Therapeutic domain of Engineered pembrolizumab designs 14c (SEQ ID NO: 127 )

[0258] QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRV TLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRST SESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYT

[0259] In some embodiments, the therapeutic domain provided herein comprise a M5A-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0260] Therapeutic domain of Engineered M5A design 15 (SEQ ID NO: 24)

[0261] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYMHWVRQAPGKGLEWVARIDPANGNSKYADSVKGRF TISADTSKNTAYLQMNSLRAEDTAVYYCAPFGYYVSDYAMAYWGQGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNH KPSNTKVDKKVEPKSC In some embodiments, the therapeutic domain provided herein comprise an ipilimumab-derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0262] Therapeutic domain of Engineered ipilimumab design 16 (SEQ ID NO: 128 )

[0263] QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFT ISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNT KVDKRVEPKSC

[0264] In some embodiments, the therapeutic domain provided herein comprise a trastuzumab- derived fragment that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to the sequence:

[0265] Therapeutic domain of Engineered PEGylated trastuzumab scFv design 20 (SEQ ID NO: 100) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSD IQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0266] Suitably, the cleavable domain does not (significantly) interfere with the binding of the agent to its binding partner. It will be appreciated that the cleavable domain is positioned in the agent such that, in a cleaved state (therapeutic domain) or uncleaved state (agent), the ability of the agent to bind to its binding partner is not (significantly) affected by the cleavable domain. Those skilled in the art would be capable of selecting an appropriate method to measure the ability of the agent to bind to its binding partner, for example by an ELISA or BIAcore.

[0267] In another embodiment the therapeutic domain binds to a binding partner when the cleavable domain has not been cleaved. In other words, it is not necessary for the therapeutic domain to be released from the agent for it to bind to its binding partner. In one embodiment, the therapeutic domain is not masked. By 'masked', we include the meaning that the therapeutic domain is blocked from binding to its binding partner (i.e. does not (significantly) bind its binding partner). Upon cleavage of a cleavable domain, a masked therapeutic domain would become unmasked. In another embodiment, the agent does not comprise a masking domain.

[0268] In a further embodiment, the therapeutic domain retains its ability to bind to its binding partner once the cleavable domain has been cleaved. It will be appreciated that the release of the stabilising domain from the therapeutic domain does not negatively decrease the binding of the therapeutic domain to its target. In one embodiment, the therapeutic domain binds its target with a half maximal inhibitory concentration (IC50) of from 0.2 to 1.4 nanomolar (nM) relative to its target, such as from about 0.2, 0.205, 0.210, 0.215, 0.220, 0.225, 0.230, 0.235, 0.240, 0.245, 0.250, 0.255, 0.260, 0.265, 0.270, 0.275, 0.276, 0.277, 0.278, 0.279 or 0.280 nM to about 0.260, 0.265, 0.270, 0.275, 0.280, 0.285, 0.290, 0.295, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2^ 1.325, 1.350, 1.375, 1.380, 1.385, 1.39, 1.395 or 1.4. The IC50 indicates the potency of the therapeutic domain in inhibiting a specific biological or biochemical function. It is a quantitative measure that indicates how much of a particular inhibitory substance is needed to inhibit a given biological process or biological component by 50%. Any suitable means can be used to measure ICso for example, by functional assays or with competition binding assay (such as an enzyme- linked immunosorbent assay (ELISA)). In one embodiment the IC50 is measured with an in vitro ELISA assay.

[0269] In one embodiment, the therapeutic domain has a dissociation constant (KD) of from 1.5 to 2.25 nM, such as from about 1.50, 1.51, 1.52, 1.53, 1.54, 1.55, 1.56, 1.57, 1.58, 1.59, 1.60, 1.61, 1.62, 1.63, 1.64, 1.65, 1.66, 1.67, 1.68, 1.69, 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76,

[0270] 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85, 1.86, 1.87, 1.88, 1.89 or 1.90 to about

[0271] 1.70, 1.71, 1.72, 1.73, 1.74, 1.75, 1.76, 1.77, 1.78, 1.79, 1.80, 1.81, 1.82, 1.83, 1.84, 1.85,

[0272] 1.86, 1.87, 1.88, 1.89, 1.90, 1.91, 1.92, 1.93, 1.94, 1.95, 1.96, 1.97, 1.98, 1.99, 2.00, 2.01,

[0273] 2.02, 2.03, 2.04, 2.05, 2.06, 2.07, 2.08, 2.09, 2.10, 2.11, 2.12, 2.13, 2.14, 2.15, 2.16, 2.17,

[0274] 2.18, 2.19, 2.20, 2.21, 2.22, 2.23, 2.24 or 2.25. The KD indicates the equilibrium constant that measures the propensity of one binding domain to dissociate from its target i.e., the binding domain of the therapeutic domain to its binding partner (e.g., a tumour antigen). The smaller the dissociation constant, the more tightly bound the binding domain is, or the higher the affinity between binding domain and its target. Any suitable means can be used to measure KD for example, by functional assays, with competition binding assay or with live or real time binding (such as an BIAcore). In one embodiment the KD is measured with an in vitro BIAcore assay.

[0275] In one embodiment, the cleavable domain does not (significantly) interfere with the thermal stability of the agent. It will be appreciated that the cleavable domain is positioned in the agent such that, in a cleaved state (therapeutic domain) or uncleaved state (agent), the thermal stability of the agent is not significantly affected by the cleavable domain. Those skilled in the art would be capable of selecting an appropriate method to measure thermal stability, for example fluorescence, static light scattering (SLS) and dynamic light scattering (DLS).

[0276] In one embodiment, the agent has a melting temperature (Tm) of from 65 degrees centigrade (°C) to 75°C, such as from 65.1, 65.2, 65.3, 65.4, 65.5, 65.6, 65.7, 65.8, 65.9, 66.0, 66.1, 66.2, 66.3, 66.4, 66.5, 66.6, 66.7, 66.8, 66.9, 67.0, 67.1, 67.2, 67.3, 67.4, 67.5, 67.6, 67.7, 67.8, 67.9, 68.0, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69.0, 69.1, 69.2, 69.3,

[0277] 69.4, 69.5, 69.6, 69.7, 69.8, 69.9 or 70.0°C to about 68.0, 68.1, 68.2, 68.3, 68.4, 68.5, 68.6,

[0278] 68.7, 68.8, 68.9, 69.0, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6, 69.7, 69.8, 69.9, 70.0, 70.1, 70.2,

[0279] 70.3, 70.4, 70.5, 70.6, 70.7, 70.8, 70.9, 71.0, 71.1, 71.2, 71.3, 71.4, 71.5, 71.6, 71.7, 71.8,

[0280] 71.9, 72.0, 72.1, 72.2, 72.3, 72.4, 72.5, 72.6, 72.7, 72.8, 72.9, 73.0, 73.1, 73.2, 73.3, 73.4,

[0281] 73.5, 73.6, 73.7, 73.8, 73.9, 74.0, 74.1, 74.2, 74.3, 74.4, 74.5, 74.6, 74.7, 74.8, 74.9, or 75.0°C. The Tm of a protein relates to the result of denaturation of the protein. Methods of measuring melting temperatures are well known in the art and include differential scanning calorimetry (DSC), differential scanning fluorometry (DSF) and other well-known thermal shift assays. In one embodiment the melting temperature is measured with an in vitro DSC assay.

[0282] In one embodiment, the agent has an aggregation temperature (Tagg) of from 65°C to 80°C, such as from 65.1, 65.2, 65.3, 65.4, 65.5, 65.6, 65.7, 65.8, 65.9, 66.0, 66.1, 66.2, 66.3, 66.4,

[0283] 66.5, 66.6, 66.7, 66.8, 66.9, 67.0, 67.1, 67.2, 67.3, 67.4, 67.5, 67.6, 67.7, 67.8, 67.9, 68.0,

[0284] 68.1, 68.2, 68.3, 68.4, 68.5, 68.6, 68.7, 68.8, 68.9, 69.0, 69.1, 69.2, 69.3, 69.4, 69.5, 69.6,

[0285] 69.7, 69.8, 69.9, 70.0, 70.1, 70.2, 70.3, 70.4, 70.5, 70.6, 70.7, 70.8, 70.9, 71.0, 71.1, 71.2,

[0286] 71.3, 71.4, 71.5, 72.0, 72.5, 73.0, 73.5, 74.0, 74.5 or 75.0, 75.5, 76.0, 76.5 or 77.0°C to about

[0287] 73.0, 73.5, 74.0, 74.5, 75.0, 75.5, 76.0, 76.5, 77.0, 77.5, 78.0, 78.5, 79.0, 79.1, 79.2, 79.3,

[0288] 79.4, 79.5, 79.6, 79.7, 79.8, 79.9 or 80.0°C. By "Tagg", we include the meaning of the temperature at which the onset of aggregation occurs or the temperature at which molecules have a tendency to aggregate together. Methods of measuring thermal stability are well known in the art and include dynamic light scattering (DLS), static light scattering (SLS) and / or fluorescence. In one embodiment the Taggis measured with an in vitro DLS assay.

[0289] DLS measures the hydrodynamic size and size distribution of particles in solution and can be plotted over time and temperature. In general, at low temperatures a protein may be stable and show repeatable size (and scattering intensity) measurements, whereas typically at more elevated temperatures (Tagg), protein molecules will show a tendency to aggregate.

[0290] In one embodiment, the cleavable domain does not (significantly) interfere with the binding of the stabilisation domain to Fc gamma receptors (FcRs), optionally wherein the Fc gamma receptors is Fc gamma receptor la and / or Fc gamma receptor Ila. FcRs are membrane proteins expressed by several hematopoietic cells that recognise the Fc region of several immunoglobulin classes and subclasses. The Fc region of an antibody can bind to Fc receptors (FcyRI, FcyRII, FcyRIII) expressed on the surface of immune cells, complement (Clq) and FcRn (neonatal FcR) in the blood, thereby activating the immune system. The interaction mediated by the antibody Fc domain can strongly influence the functional outcome of antibody therapy including antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell- mediated phagocytosis (ADCP) and complement-dependent cytotoxicity (CDC) through the interaction of the Fc domain with Fc receptors on different cell types. Of note, the combination of IgG and neonatal FcR (FcRn) can protect antibodies from being degraded, thereby prolonging its half-life.

[0291] In another embodiment, the cleavable domain may interfere with the binding of the stabilisation domain to Fc gamma receptors.

[0292] In one embodiment, insertion of a glycosylation site increases binding of the Fc region of the agent. One aspect of the invention therefore provides a method of increasing Fc receptor binding of an agent by insertion of a glycosylation site, preferably wherein the glycosylation site is inserted into the cleavable domain. Another aspect of the invention provides the use of a glycosylation site for increasing Fc receptor binding of the agent, preferably wherein the glycosylation site is inserted into the cleavable domain of the agent. The agent of the method or use may be as described herein according to any other aspects of the invention.

[0293] Suitable cleavable domains may display one or more N-glycosylation motifs. N-glycosylation is the process of attachment of a glycan oligosaccharide to an amide nitrogen of an asparagine (N) residue of a protein.

[0294] It will be understood by those in the art that glycosylation is the addition of carbohydrate chains (glycans) proteins. Glycosylation may be O-glycosylation or N-glycosylation. Preferably, the glycosylation site in the cleavable domain is an N-glycosylation motif. In one embodiment, the N-glycosylation motif is located after the cysteine residue that ends the CHI domain, optionally prior to the hinge disulphide bond region of the agent, for example in the YNSTY (SEQ ID NO: 126) sequence of the CH2 of an Fc region, for example N297.

[0295] It will be appreciated that the cleavable domain is positioned in the agent such that, in a cleaved state (stability domain) or uncleaved state (agent), the binding of Fc gamma receptors is not significantly affected by the cleavable domain. Those skilled in the art would be capable of selecting an appropriate method to measure binding, for example by functional assays or with competition binding assay (such as an enzyme-linked immunosorbent assay (ELISA)).

[0296] In one embodiment, the therapeutic domain reduces or inhibits the proliferation of unwanted cells or the growth of a tumour.

[0297] In one embodiment, the therapeutic domain (for example when bound to its target or binding partner) stimulates immune cells, for example immune checkpoint inhibitors (ICIs), immunomodulators, cytokines.

[0298] It will be appreciated that an immune cell may comprise any of the following immune cell: lymphocytes (B and T cells), antigen presenting cells (APC), natural killer (NK) cells, macrophages, monocytes, dendritic cells. T cells recognise peptide antigens, derived from proteins degraded intracellularly, that are loaded onto cell surface MHC molecules, a process called antigen presentation (APC).

[0299] By "stimulation", we include the meaning that an immune cell, such as a T cell, is activated and proliferates. In some embodiments, stimulation also includes co-stimulation, preventing recruitment of inhibitory effectors and preventing T cell exhaustion.

[0300] In another embodiment, the therapeutic domain blocks immune cells. The terms "blocks" and "inhibits" are used interchangeably and encompass both partial and complete inhibition / blocking. In one embodiment the blocked immune cells are regulatoryT cells (Tregs). Tregs are a specialised subpopulation of T cells that play a critical role in preventing autoimmunity, by inhibiting T cell proliferation and cytokine production.

[0301] As discussed herein, the primary function of the therapeutic domain is to exert a clinical or therapeutically beneficial effect on the target tissue. Optionally, the therapeutic domain may have a secondary function wherein the therapeutic domain targets (e.g. localises) the agent to the target tissue. For example, wherein the therapeutic domain according to the claimed invention is an antibody drug conjugate (ADC), it may comprise a Fab domain (which targets the ADC to the unwanted cells), and a cytotoxic drug (also known as the payload) chemically linked to a Fab region. Therefore, the therapeutic domain may function to (i) exert a clinical or therapeutically beneficial effect and / or (ii) target itself (or the agent) to the target tissue.

[0302] Stabilisation domain

[0303] In some embodiments, the stabilisation domain is a protein-based domain or a polymer. For example, the protein-based domain may comprise a structured polypeptide (e.g. an IgG Fc region, HSA), an elastin-like peptide (ELPylationn), an inert polypeptide, e.g., XTEN (also known as recombinant PEG or"rPEG"), a homoamino acid polymer (HAP; HAPylation) proline-alanine- serine polymer (PAS; PASylation). In another embodiment, the stabilisation domain is not a protein, for example the stabilisation domain is a chemical moiety (e.g. PEGylation or hyaluronic acid).

[0304] In one embodiment, the stabilisation domain targets the therapeutic domain to unwanted cells. By "stabilisation domain targeting the therapeutic domain", we include the meaning that that stabilisation domain may by a specific binding partner of an entity expressed by or associated with unwanted cells. In another embodiment, the stabilisation domain does not target the therapeutic domain to the unwanted cells.

[0305] In some embodiments, the stabilisation domain is: i. a Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region; or; or ii. a PEGylated domain; or iii. a PASylation domain; or iv. a XTENylated domain; or v. a HESylated domain; or v. a lipidated domain; or vii. a glycosylated domain; or viii. the Human Serum Albumin (HSA) protein or fragment thereof; or ix. a HSA binding protein; or x. a protein binding to a blood circulating cell or xi. a Fab domain, optionally wherein the Fab domain is directed HSA or directed to the same antigen as the antigen-binding domain. Preferably, the stabilisation domain is: i. a Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region. In one embodiment, the stabilisation domain does not comprise a Fab domain.

[0306] In some embodiments, the stabilisation domain comprises or consists of an Fc region, which may or may not include all or part (e.g. all or part of the lower and / or middle) of the hinge region. The Fc region and / or the hinge region (or any part thereof) may be selected from any type of immunoglobulins which includes IgM, IgG, IgA, IgD, IgE, IgY. Each isotype also includes different subtypes. For example, subtypes of IgG include IgGl, IgG2, IgG3, IgG4 and subtypes of IgA include IgAl and IgA2. It will be appreciated that there are different isotypes and subtypes in different species. The Fc region and / or the hinge region can be of any class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, in particular IgGl or IgG4), or any subclass (e.g., IgG2a and IgG2b) of immunoglobulin molecule.

[0307] Preferably, the agent is based on an antibody which is human or humanised. In certain embodiments, agents of the invention are based on IgG antibodies (or all or part of an Fc region for the stabilisation domain), or a class (e.g., human IgGl or IgG4) or subclass thereof. In certain embodiments, the agents of the invention are based on an antibody which comprises or comprise a stabilisation domain which comprises (all or part of) a human gamma 4 constant region. In another embodiment, the heavy chain constant region (e.g. an Fc domain of the stabilisation domain) does not bind Fc-y receptors, and e.g. comprises a Leu235Glu mutation (EU numbering). In another embodiment, the heavy chain constant region (e.g. an Fc domain of the stabilisation domain) comprises a Ser228Pro mutation to increase stability. In another embodiment, the heavy chain constant region (e.g. an Fc domain of the stabilisation domain) is IgG4-PE.

[0308] In a preferred embodiment, the stabilisation domain comprises or consists of an Fc region in (or derived from) an IgGl or IgG4 subclass. The Fc region of the stabilisation domain may be a homodimeric structure comprising a covalent linkage via two disulphide bonds located in the middle hinge region (via cysteine residues).

[0309] IgGl Fc regions of the stabilisation domain may comprise cysteine residues located at positions 11 and 14 (IMGT hinge numbering scheme), positions 226 and 229 (EU numbering scheme), positions 239 and 242 (Kabat numbering) and / or positions 239 and 242 (Chothia numbering); these cysteines are used to form disulphide bridges to covalently link the two polypeptide chains in the homodimer. In other embodiments, the cleavage domain is located after the cysteine residues located at positions 11 and 14 (IMGT hinge numbering scheme), positions 226 and 229 (EU numbering scheme), positions 239 and 242 (Kabat numbering) and / or positions 239 and 242 (Chothia numbering), such that the stabilisation domain, when cleaved, does not comprise these residues.

[0310] IgGl Fc regions of the stabilisation domain may also comprise proline residues at positions 12 and 13 (IMGT hinge numbering scheme), positions 227 and 228 (EU numbering scheme), positions 240 and 241 (Kabat numbering) and / or positions 240 and 241 (Chothia numbering). In other embodiments, the cleavage domain is located after the proline residues at positions 12 and 13 (IMGT hinge numbering scheme), positions 227 and 228 (EU numbering scheme), positions 240 and 241 (Kabat numbering) and / or positions 240 and 241 (Chothia numbering), such that the stabilisation domain, when cleaved, does not comprise these residues.

[0311] Preferably the cysteine and / or proline residues in these positions are left intact (i.e. not mutated or changed) in the agent (i.e. whether part of the stabilisation domain, cleavage domain or therapeutic domain).

[0312] IgG4 Fc regions of the stabilisation domain may comprise cysteine residues located in positions 8 and 11 (IMGT hinge numbering scheme), positions 226 and 229 (EU numbering), positions

[0313] 239 and 242 (Kabat numbering) and / or positions 239 and 242 (Chothia numbering); these cysteines are used to form disulphide bridges to covalently link the two polypeptide chains in the homodimer. In other embodiments, the cleavage domain is located after the cysteine residues located at positions 8 and 11 (IMGT hinge numbering scheme), positions 226 and 229 (EU numbering), positions 239 and 242 (Kabat numbering) and / or positions 239 and 242 (Chothia numbering), such that the stabilisation domain, when cleaved, does not comprise these residues.

[0314] Preferably, IgG4 Fc regions may comprise a proline and a serine residue at positions 9 and 10 respectively (IMGT hinge numbering scheme), positions 227 and 228 (EU numbering), positions

[0315] 240 and 241 (Kabat numbering) and / or positions 240 and 241 (Chothia numbering). In other embodiments, the cleavage domain is located after the proline and serine residues located at positions 9 and 10 (IMGT hinge numbering scheme), positions 227 and 228 (EU numbering), positions 240 and 241 (Kabat numbering) and / or positions 240 and 241 (Chothia numbering) such that the stabilisation domain, when cleaved, does not comprise these residues.

[0316] In some alternative embodiments, the IgG4 Fc regions may be a modified IgG4 Fc region which comprises proline residues at positions 9 and 10 (IMGT hinge numbering scheme), positions 1 7 and 228 (EU numbering), positions 240 and 241 (Kabat numbering) and / or positions 240 and 241 (Chothia numbering). In other embodiments, the cleavage domain is located after the proline residues located at positions 9 and 10 (IMGT hinge numbering scheme), positions 227 and 228 (EU numbering), positions 240 and 241 (Kabat numbering) and / or positions 240 and 241 (Chothia numbering) such that the stabilisation domain, when cleaved, does not comprise these residues. In the modified IgG4, the serine at position 10 is substituted for a proline in order to improve structural rigidity.

[0317] Preferably the cysteine and / or proline and / or serine residues in these positions are left intact (i.e. not mutated or changed) in the agent (i.e. whether part of the stabilisation domain, cleavage domain or therapeutic domain).

[0318] These Fc regions may comprise a mutation that add extra functionality or stability to the Fc domains. The Fc region may comprise two or more mutations that add extra functionality or stability to the Fc region, e.g. 2, 3, 4 or 5 mutations. The skilled person would be able to select an appropriate mutation from those known in the art. Examples of such mutations are (but not limited to):

[0319] • N297A mutation (EU numbering);

[0320] • S224P mutations (EU numbering) and / or L235E (EU numbering) [preferably in IgG4 Fc regions];

[0321] • M252Y / S254T / T256E (EU numbering schemes;

[0322] • T366W + T366S / L368A / Y407V mutations (EU numbering schemes) [preferably in IgGl Fc regions];

[0323] • T250Q / M428L (EU numbering); and

[0324] • H433K / N434F (EU numbering).

[0325] N297A mutations reduce Fc gamma receptor binding. S224P mutations (in IgG4 Fc regions) prevent interchain strand exchange. M252Y / S254T / T256E mutations in IgGl may extend the half-life. T366W in combination with T366S / L368A / Y407V mutations (in IgGl Fc regions) encourage heterodimerisation, e.g. for the making of bispecific antibody molecules. T250Q / M428L and H433K / N434F may increase the half-life of IgGl Fc regions and / or increase the binding affinity to FcRn.

[0326] For IgGl, the Fc region of the stabilisation domain may start at positions 6 to 10 (IMGT hinge numbering), positions 221 to 225 (EU numbering), positions 234-238 (Kabat numbering) and / or positions 234-238 (Chothia numbering). For IgG4, the Fc region of the stabilisation domain may start at positions 5 to 7 (IMGT hinge numbering), positions 218 to 225 (EU numbering), positions 230 to 238 (Kabat numbering) and / or positions 230 to 238 (Chothia numbering). The Fc region of the stabilisation domain may end at positions 125 to 130 (IMGT C-domain numbering for CH3), positions 445 to 447 (EU numbering), positions 476 to 478 (Kabat numbering) and / or (Chothia numbering).

[0327] Other serum proteins that may act as stabilisation domain may include albumin, fibrinogen, fibronectin, haemoglobin, transferrin, an immunoglobulin domain.

[0328] Suitable stabilisation domains may display one or more N-glycosylation motifs. N-glycosylation is the process of attachment of a glycan oligosaccharide to an amide nitrogen of an asparagine (N) residue of a protein. N-glycosylation motifs can be found in the YNSTY (SEQ ID NO: 126) sequence of the CH2 of an Fc region, for example N297.

[0329] The Fc region contains the constant regions (CH) CH2, and CH3 from the heavy chains. The general shape of an antibody is a Y, with a flexible hinge (interdomain) region at the centre of the Y. The flexibility of the interdomain hinge region is important for the bivalent binding of an antibody, allowing the two binding pockets to interact with antigenic sites at variable distances. The Fc region contains the constant regions (CH) CH2, and CH3 from the heavy chains. The Fc region may be composed of homo-immunoglobulin molecules, preferably homo-IgG molecules. In one embodiment, the stabilisation domain is not a hetero-IgG molecule.

[0330] In some embodiments, the stabilisation domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to sequences selected from the group consisting of:

[0331] Stabilisation domain of Engineered trastuzumab designs 1-6, Dlx-6x, D9, D12, D13, D15 and D18 (SEQ ID NO: 27) ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL SPGK

[0332] Stabilisation domain of Engineered trastuzumab design 7 (SEQ ID NO: 28)

[0333] FLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLH QDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEW ESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0334] Stabilisation domain of Engineered pembrolizumab designs D14a, D14b and D14c (SEQ ID NO: 29) EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRV VSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGF YPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSL SLSLGK

[0335] Stabilisation domain of Engineered pembrolizumab designs D14c (SEQ ID NO: 101)

[0336] NVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQ FNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQP REPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDK SRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK

[0337] For design 20, the stabilisation domain comprises or consists of a polyethylene glycol (PEG) polymer.

[0338] As discussed herein, the primary function of the stabilisation domain is to stabilise or to extend the half-life of the agent in a biological system. Optionally, the stabilisation domain may have a secondary function wherein the stabilisation domain has a clinical or therapeutically beneficial effect. For example, wherein the agent according to the claimed invention is an antibody drug conjugate (ADC), it may comprise a therapeutic domain (which targets the ADC to the unwanted cells), a cleavable domain and a stabilisation domain. The stabilisation domain may comprise a cytotoxic drug (also known as the payload) chemically linked to an Fc region. Therefore, the stabilisation domain may function to (i) stabilise or increase the half-life of the agent and (ii) exert a clinical or therapeutically beneficial effect.

[0339] Further, Fc regions have been engineered to include antigen-binding domains in the CH3 domains, known as Fcabs, see for example W02006 / 072620 and WO2009 / 132876 (both F-Star Biotechnologische Forschungs und Entwicklungsges M.B.H). Thus, the clinical or therapeutically beneficial effect of the Fc domain may be due to the presence of an antigen-binding domain which has been engineered into the Fc domain. Thus, in one embodiment, the Fc domain comprises or consists of an Fcab.

[0340] Cleavable domain

[0341] The cleavable domain is positioned between the therapeutic domain and the stabilisation domain and connects the therapeutic and stabilisation domains in the agent. Selective cleavage of the cleavable domain releases the therapeutic domain from the stabilisation domain enabling the therapeutic domain to carry out its therapeutic function. Typically, the stabilisation domain will then be degraded or released and cleared by GFB. It will be appreciated that when the agent is cleaved, part of the cleavable domain may remain attached to the therapeutic domain, and part of the cleavable domain may remain attached to the stabilisation domain. The cleavable domain comprises at least one cleavage site. In one embodiment, the cleavable domain consists of the cleavage site. "Cleavage site" refers to a site of an amino acid sequence that is a substrate for an enzyme, such as an extracellular enzyme. For example, the cleavage site for the enzyme urokinase-type plasminogen activator (uPA), fibroblast activation protein (FAP), legumain, metalloproteases or MT-SP1. The cleavage site may be one that is cleavable by an enzyme such as any of a protease, a nuclease, a lipase, a lyase, a phosphatase or a carbohydrase, which may or may not be membrane-bound. By "protease", we include the meaning of an enzyme that catalyses proteolysis (i.e. the breaking down of a protein into smaller polypeptides or single amino acids). In one embodiment, the cleavage site is not the amino acid sequence DEVD (SEQ ID NO: 151).

[0342] It will be appreciated that the cleavable domain may or may not comprise a hinge region or part thereof. The cleavable domain may comprise a cleavage site and linker domain sequence(s) which connect the therapeutic domain (such as OmoMYC) to a stabilisation domain, wherein the stabilisation domain is not an Fc region or part thereof (such as HSA).

[0343] In a particular embodiment, the cleavage site is located in a hinge region. By "a hinge region", we include the meaning of a hydrophilic sequence of the heavy chains of an antibody. In general, the hinge is responsible for linking a Fab region to an Fc region in a flexible manner. The hinge can be divided into three parts: the upper hinge, middle hinge, and lower hinge. The middle hinge is where the two heavy chains meet, and this is the part that holds the antibody together.

[0344] In a particular embodiment, the cleavable domain is located in the upper hinge, middle hinge, or lower hinge, preferably in the upper hinge or in the lower hinge. Advantages of the cleavable domain in the hinge region include providing the ability of fully separating the therapeutic and stabilisation domains after cleavage.

[0345] In a preferred embodiment, the cleavable domain is located in the lower hinge. A potential benefit of this approach is that it ensures that the payload is co-located with the therapeutic domain following cleavage of the cleavable domain, for example in instances where the conjugation site is located in the middle hinge (such as when the conjugation site is a cysteine residue in the middle hinge region).

[0346] In an alternative embodiment, the cleavable domain is located in the upper hinge.

[0347] Preferably the residues are substituted for residues in the cleavable domain, such as residues in the cleavage site and / or linker domain(s). The cleavable domain may be introduced into the hinge region of an antibody (e.g. any of the antibodies described herein, such as Trastuzumab or Pembrolizumab).

[0348] The cleavable domain may be introduced into the hinge region of an IgGl antibody (e.g. Trastuzumab). Structurally, this is defined in IgGl type antibodies as:

[0349] Table A:

[0350] IgGl upper and middle hinge region (SEQ ID NO: 154): EPKSCDKTHTCPPCP

[0351] Table B:

[0352] IgGl lower hinge region (SEQ ID NO: 155): (A)PELLGGPSVFLF

[0353] The cleavable domain and / or the cleavage site may be introduced into the hinge region of an IgG4 antibody (e.g. Pembrolizumab). Structurally, this is defined in IgG4 type antibodies as: Table C: IgG4 upper and middle hinge region (SEQ ID NO: 156): (E)SKYGPPCPSCP

[0354] Table D: IgG4 lower hinge region (SEQ ID NO: 157): (A)PEFLGGPSVFLF

[0355] The cleavable domain may be engineered into the hinge region as an insertion, substitution or a combination thereof. The total insertion may be between 1 and 30, 2 and 26, 3 and 26, 3 and 20, 3 and 15, 3 and 8 or 3 and 5 amino acids in length, and is preferably between 3 and 26 amino acids in length (inclusive). The insertion may be up to 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,

[0356] 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids in length. Preferably, the insertion is between 3 and 26 amino acids in length. A maximum of 10 (for example 9, 8, 7, 6, 5, 4, 3, 2 or 1, in particular 5 or fewer) amino acids of the hinge region may be deleted and replaced with amino acids of the cleavage domain.

[0357] In some embodiments, the C-terminal cysteine of the CHI domain is left intact. By "intact", we include that the residue at this position is not mutated or changed from wild-type, e.g. due to the presence of the cleavable domain. For example, the C-terminal cysteine of the CHI domain is not deleted or substituted. In another embodiment, the agent comprises a C-terminal cysteine of the CHI domain. The cleavable domain and / or cleavage site may be located before or after the C-terminal cysteine of the CHI domain. Preferably, the cleavable domain or cleavage site is located after the C-terminal cysteine of the CHI domain (i.e. after the C- terminus of cysteine). By "C-terminal cysteine of the CHI domain", we include the cysteine at position 5 (IMGT hinge numbering), position 220 (EU numbering), position 233 (Kabat numbering) and / or position 233 (Chothia numbering) in an IgGl type antibody or a cysteine at the corresponding position on other antibody types. In one embodiment the cleavable domain or cleavage site is located up to 5 amino acids after the C-terminal cysteine of the CHI domain, for example 1, 2, 3, 4, or 5 amino acids (in particular 1 amino acid) after the C-terminal cysteine of the CHI domain.

[0358] Cleavable domain in upper hinge

[0359] In some embodiments, the cleavable domain and / or the cleavage site may be located in the upper hinge (for example as defined by or including positions 1-10 (IMGT IgGl hinge numbering), 216-225 (EU numbering), 226-238 (Kabat numbering) and / or 226-238 (Chothia numbering) for hinges derived from IgGl antibodies and / or positions 1-7 (IMGT IgG4 hinge numbering), 216-225 (EU numbering), 226-238 (Kabat numbering) and / or 226-238 (Chothia numbering) for hinges derived from IgG4 antibodies,). Suitably the cleavable domain and / or the cleavage site may be located before the first C of the hinge motif CPPC (or CPSC).

[0360] / . IgGl type antibodies or Fc regions derived therefrom

[0361] The cleavable domain and / or the cleavage site may be located in a hinge region of an IgGl antibody or an antibody derived therefrom, optionally the upper hinge region. The cleavable domain and / or cleavage site may be introduced into the hinge region by insertion and / or substitution.

[0362] In some embodiments, the cleavable domain and / or cleavage site is located after:

[0363] • position 1 as defined by IMGT Hinge numbering;

[0364] • position 216 as defined by EU numbering;

[0365] • position 226 as defined by Kabat numbering; and / or

[0366] • position 226 as defined by Chothia numbering. Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0367] • position 11 as defined by IMGT hinge numbering;

[0368] • position 226 as defined by EU numbering;

[0369] • position 239 as defined by Kabat numbering; and / or

[0370] • position 239 as defined by Chothia numbering.

[0371] Additionally or alternatively, the cleavable domain and / or cleavage site ends at:

[0372] • position 10 as defined by IMGT hinge numbering;

[0373] • position 225 as defined by EU numbering;

[0374] • position 238 as defined by Kabat numbering; and / or

[0375] • position 238 as defined by Chothia numbering.

[0376] In some embodiments, the cleavable domain and / or cleavage site is:

[0377] • located after position 1 and before position 11, as defined by IMGT hinge numbering;

[0378] • located after position 216 and before position 226, as defined by EU numbering;

[0379] • located after position 226 and before position 239, as defined by Kabat numbering; and / or

[0380] • located after position 226 and before position 239, as defined by Chothia numbering.

[0381] In some embodiments, the cleavable domain and / or cleavage site is:

[0382] • located after position 1 and ends at position 10, as defined by IMGT hinge numbering;

[0383] • located after position 216 and ends at position 225, as defined by EU numbering;

[0384] • located after position 226 and ends at position 238, as defined by Kabat numbering; and / or

[0385] • located after position 226 and ends position 238, as defined by Chothia numbering.

[0386] By "located" we include that the cleavable domain and / or cleavage site is present at this location in the hinge. For example, the cleavable domain and / or cleavage site may be inserted at this position and / or substituted with relevant amino acids. The intervening amino acids between the positions in the hinge sequence may be maintained, substituted or deleted.

[0387] By "located before" position Z, we include that the cleavable domain and / or cleavage site is located prior to the N-terminus of the residue at position Z.

[0388] By "located after" position X, we mean that the cleavable domain and / or cleavage site is located after the C-terminus of the residue in position X, e.g. the N-terminal amino acid of the cleavable domain and / or cleavage site is located after the C-terminus of the residue in position X. The cleavable domain and / or cleavage site may begin at the position that immediately follows positions X, e.g. at position X+l. The term includes the introduction of the cleavable domain or cleavage site as an insertion and / or substitution immediately following position X, but does not include a substitution at position X itself.

[0389] By "ends at" position Y, we mean that the end of the cleavable domain and / or cleavage site terminates at or before the N-terminus of the amino acid in position Y+ l, e.g. the C-terminal amino acid of the cleavable domain and / or cleavage site is located before the N-terminus of the residue in position Y+ l. The term would encompass a substitution at position Y itself. However, where the cleavable domain and / or cleavage site is engineered into the hinge by an insertion, the insertion would need to finish prior to the residue in position Y+l, e.g. finish at position Y-l.

[0390] In an embodiment, the cleavable domain and / or cleavage site is located after:

[0391] • position 5 as defined by IMGT Hinge numbering;

[0392] • position 220 as defined by EU numbering;

[0393] • position 233 as defined by Kabat numbering; and / or

[0394] • position 233 as defined by Chothia numbering.

[0395] Additionally or alternatively, the cleavable domain and / or cleavage site is located before and / or ends at:

[0396] • position 11 as defined by IMGT hinge numbering;

[0397] • position 226 as defined by EU numbering;

[0398] • position 239 as defined by Kabat numbering; and / or

[0399] • position 239 as defined by Chothia numbering.

[0400] Additionally or alternatively, the cleavable domain and / or cleavage site ends at:

[0401] • position 10 as defined by IMGT hinge numbering;

[0402] • position 225 as defined by EU numbering;

[0403] • position 238 as defined by Kabat numbering; and / or

[0404] • position 238 as defined by Chothia numbering.

[0405] In an embodiment, the cleavable domain and / or cleavage site is:

[0406] • located after position 5 and before position 11, as defined by IMGT hinge numbering;

[0407] • located after position 220 and before position 226, as defined by EU numbering;

[0408] • located after position 233 and before position 239, as defined by Kabat numbering; and / or

[0409] • located after position 233 and before position 239, as defined by Chothia numbering.

[0410] In an embodiment, the cleavable domain and / or cleavage site is: • located after position 5 and ends at position 10, as defined by IMGT hinge numbering;

[0411] • located after position 220 and ends at position 225, as defined by EU numbering;

[0412] • located after position 233 and ends at position 238, as defined by Kabat numbering; and / or

[0413] • located after position 233 and ends at position 238, as defined by Chothia numbering.

[0414] In an embodiment, the residues at the following positions in the hinge are deleted and / or are substituted (in particular are substituted) by residues from the cleavable domain (e.g. the cleavage site and / or linker domain(s)):

[0415] • positions 5 to 10, as defined by IMGT hinge numbering;

[0416] • positions 220 to 225, as defined by EU numbering;

[0417] • positions 233 to 238, as defined by Kabat numbering; and / or

[0418] • positions 233 to 238, as defined by Chothia numbering.

[0419] In a preferred embodiment, the cleavable domain and / or cleavage site is:

[0420] • located after position 5 and is located before position 11, as defined by IMGT unique numbering for C-domain;

[0421] • located after position 220 and is located before position 226, as defined by EU numbering;

[0422] • located after position 233 and is located before position 239, as defined by Kabat numbering; and / or

[0423] • located after position 233 and is located before position 239, as defined by Chothia numbering, and the residues in the following positions of the hinge are deleted:

[0424] • positions 6-10 (inclusive) as defined by IMGT hinge numbering;

[0425] • positions 221-225 (inclusive) as defined by EU numbering;

[0426] • positions 234-238 (inclusive) as defined by Kabat numbering; and / or

[0427] • positions 234-238 (inclusive) as defined by Chothia numbering.

[0428] The cleavable domain and / or cleavage site may be:

[0429] • located after position 5 and ends by position 10, as defined by IMGT unique numbering for C-domain;

[0430] • located after position 220 and ends by position 225, as defined by EU numbering;

[0431] • located after position 233 and ends by position 238, as defined by Kabat numbering; and / or

[0432] • located after position 233 and ends by position 238, as defined by Chothia numbering. and the residues in the following positions of the hinge are deleted: • positions 6-10 (inclusive) as defined by IMGT hinge numbering;

[0433] • positions 221-225 (inclusive) as defined by EU numbering;

[0434] • positions 234-238 (inclusive) as defined by Kabat numbering; and / or

[0435] • positions 234-238 (inclusive) as defined by Chothia numbering.

[0436] In a preferred embodiment, the cleavable domain and / or cleavage site is:

[0437] • located after position 5 and is located before position 11, as defined by IMGT unique numbering for C-domain;

[0438] • located after position 220 and is located before position 226, as defined by EU numbering;

[0439] • located after position 233 and is located before position 239, as defined by Kabat numbering; and / or

[0440] • located after position 233 and is located before position 239, as defined by Chothia numbering, and the residues in the following positions of the hinge are substituted with residues from the cleavable domain (e.g. the cleavage site and / or linker domain(s)):

[0441] • positions 6-10 (inclusive) as defined by IMGT hinge numbering;

[0442] • positions 221-225 (inclusive) as defined by EU numbering;

[0443] • positions 234-238 (inclusive) as defined by Kabat numbering; and / or

[0444] • positions 234-238 (inclusive) as defined by Chothia numbering.

[0445] The cleavable domain and / or cleavage site may be:

[0446] • located after position 5 and ends by position 10, as defined by IMGT unique numbering for C-domain;

[0447] • located after position 220 and ends by position 225, as defined by EU numbering;

[0448] • located after position 233 and ends by position 238, as defined by Kabat numbering; and / or

[0449] • located after position 233 and ends by position 238, as defined by Chothia numbering. and the residues in the following positions of the hinge are substituted with residues from the cleavable domain (e.g. the cleavage site and / or linker domain(s)):

[0450] • positions 6-10 (inclusive) as defined by IMGT hinge numbering;

[0451] • positions 221-225 (inclusive) as defined by EU numbering;

[0452] • positions 234-238 (inclusive) as defined by Kabat numbering; and / or

[0453] • positions 234-238 (inclusive) as defined by Chothia numbering.

[0454] In a preferred embodiment, the cleavable domain or cleavage site is:

[0455] • located between positions 5 and 11, as defined by IMGT Hinge numbering;

[0456] • located between positions 220 and 226, as defined by EU numbering;

[0457] • located between positions 233 and 239, as defined by Kabat numbering; and / or located between positions 233 and 239, as defined by Chothia numbering.

[0458] In a preferred embodiment, the cleavable domain and / or cleavage site is located after:

[0459] • position 5 as defined by IMGT Hinge numbering;

[0460] • position 220 as defined by EU numbering;

[0461] • position 233 as defined by Kabat numbering; and / or

[0462] • position 233 as defined by Chothia numbering.

[0463] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0464] • position 6 as defined by IMGT Hinge numbering;

[0465] • position 221 as defined by EU numbering;

[0466] • position 234 as defined by Kabat numbering; and / or

[0467] • position 234 as defined by Chothia numbering.

[0468] Additionally or alternatively, the cleavable domain and / or cleavage site ends at:

[0469] • position 6 as defined by IMGT Hinge numbering;

[0470] • position 221 as defined by EU numbering;

[0471] • position 234 as defined by Kabat numbering; and / or

[0472] • position 234 as defined by Chothia numbering.

[0473] In a preferred embodiment, the cleavable domain and / or cleavage site is:

[0474] • located after position 5 and is located before position 6, as defined by IMGT unique numbering for C-domain;

[0475] • located after position 220 and is located before position 221, as defined by EU numbering;

[0476] • located after position 233 and is located before position 234, as defined by Kabat numbering; and / or

[0477] • located after position 233 and is located before position 234, as defined by Chothia numbering.

[0478] In a preferred embodiment, the cleavable domain and / or cleavage site is:

[0479] • located after position 5 and ends by position 6, as defined by IMGT unique numbering for C-domain;

[0480] • located after position 220 and ends by position 221, as defined by EU numbering;

[0481] • located after position 233 and ends by position 234, as defined by Kabat numbering; and / or

[0482] • located after position 233 and ends by position 234, as defined by Chothia numbering. The cleavable domain or cleavage site may be engineered into the hinge as an insertion between:

[0483] • positions 5 and 6, as defined by IMGT Hinge numbering;

[0484] • positions 220 and 221, as defined by EU numbering;

[0485] • positions 233 and 234, as defined by Kabat numbering; and / or

[0486] • positions 233 and 234, as defined by Chothia numbering.

[0487] In an embodiment, one, two, three, four or five of the amino acids in positions 5-10 (IMGT hinge numbering), positions 220-225 (EU numbering), positions 233-238 (Kabat numbering) and / or positions 233-238 (Chothia numbering) of the hinge are deleted or substituted by the cleavable domain (e.g. the cleavage site and / or linker domain(s)).

[0488] In an embodiment, one, two, three, four or five of the amino acids in positions 5-10 (IMGT hinge numbering), positions 220-225 (EU numbering), positions 233-238 (Kabat numbering) and / or positions 233-238 (Chothia numbering) of the hinge are deleted.

[0489] In an embodiment, one, two, three, four or five of the amino acids in positions 5-10 (IMGT hinge numbering), positions 220-225 (EU numbering), positions 233-238 (Kabat numbering) and / or positions 233-238 (Chothia numbering) of the hinge are substituted by the cleavable domain (e.g. the cleavage site and / or linker domain(s)).

[0490] In an embodiment, the residues in one or more of the following positions are left intact:

[0491] • position 5 (IMGT hinge numbering IgGl), 220 (EU numbering), 233 (Kabat numbering) and / or 233 (Chothia numbering); and / or

[0492] • position 11 (IMGT hinge numbering IgGl), 226 (EU numbering), 239 (Kabat numbering) and / or 239 (Chothia numbering); and / or

[0493] • position 12 (IMGT hinge numbering IgGl), 227 (EU numbering), 240 (Kabat numbering) and / or 240 (Chothia numbering); and / or

[0494] • position 13 (IMGT hinge numbering IgGl), 228 (EU numbering), 241 (Kabat numbering) and / or 241 (Chothia numbering); and / or

[0495] • position 14 (IMGT hinge numbering IgGl), 229 (EU numbering), 242 (Kabat numbering) and / or 242 (Chothia numbering); and / or

[0496] • position 15 (IMGT hinge numbering IgGl), 230 (EU numbering), 243 (Kabat numbering) and / or 243 (Chothia numbering).

[0497] Optionally, position 1.6 (IMGT unique numbering for C-domain CH2), 231 (EU numbering), 244 (Kabat numbering) and / or 244 (Chothia numbering) may be left intact. By "intact", we include that the residue at this position is not mutated or changed, e.g. due to the presence of the cleavable domain and / or cleavage site. In another embodiment, the agent comprises (native) amino acid residues at one or more (e.g. all) of the above positions.

[0498] / / . IgG4 type antibodies or Fc regions derived therefrom

[0499] The cleavable domain and / or the cleavage site may be located in a hinge region of an IgG4 antibody or an antibody derived therefrom, optionally the upper hinge region. The cleavable domain and / or cleavage site may be introduced into the hinge region by insertion and / or substitution.

[0500] In some embodiments, the cleavable domain is located after:

[0501] • position 1 as defined by IMGT Hinge numbering;

[0502] • position 216 as defined by EU numbering;

[0503] • position 226 as defined by Kabat numbering; and / or

[0504] • position 226 as defined by Chothia numbering.

[0505] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0506] • position 8 as defined by IMGT hinge numbering;

[0507] • position 226 as defined by EU numbering;

[0508] • position 239 as defined by Kabat numbering; and / or

[0509] • position 239 as defined by Chothia numbering.

[0510] Additionally or alternatively, the cleavable domain ends at:

[0511] • position 7 as defined by IMGT hinge numbering;

[0512] • position 225 as defined by EU numbering;

[0513] • position 238 as defined by Kabat numbering; and / or

[0514] • position 238 as defined by Chothia numbering.

[0515] In some embodiments, the cleavable domain is:

[0516] • located after position 1 and before position 8, as defined by IMGT hinge numbering;

[0517] • located after position 216 and before position 226, as defined by EU numbering;

[0518] • located after position 226 and before position 239, as defined by Kabat numbering; and / or

[0519] • located after position 226 and before position 239, as defined by Chothia numbering.

[0520] In some embodiments, the cleavable domain is:

[0521] • located after position 1 and ends at position 7, as defined by IMGT hinge numbering;

[0522] • located after position 216 and ends at position 225, as defined by EU numbering; • located after position 226 and ends at position 238, as defined by Kabat numbering; and / or

[0523] • located after position 226 and ends position 238, as defined by Chothia numbering.

[0524] In some embodiments, the cleavable domain and / or cleavage site is located after:

[0525] • position 4 as defined by IMGT Hinge numbering;

[0526] • position 218 as defined by EU numbering;

[0527] • position 229 as defined by Kabat numbering; and / or

[0528] • position 229 as defined by Chothia numbering.

[0529] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0530] • position 8 as defined by IMGT hinge numbering;

[0531] • position 226 as defined by EU numbering;

[0532] • position 239 as defined by Kabat numbering; and / or

[0533] • position 239 as defined by Chothia numbering.

[0534] In some embodiments, the cleavable domain and / or cleavage site is:

[0535] • located after position 4 and before position 8, as defined by IMGT hinge numbering;

[0536] • located after position 218 and before position 226, as defined by EU numbering;

[0537] • located after position 229 and before position 239, as defined by Kabat numbering; and / or

[0538] • located after position 229 and before position 239, as defined by Chothia numbering.

[0539] The cleavable domain and / or cleavage site may be inserted in the hinge region between:

[0540] • position 4 and 8, as defined by IMGT hinge numbering;

[0541] • position 218 and 226, as defined by EU numbering;

[0542] • position 229 and 239, as defined by Kabat numbering; and / or

[0543] • position 229 and 239, as defined by Chothia numbering.

[0544] In an embodiment, one, two or three of the residues in positions 5-7 (IMGT hinge numbering IgG4), positions 233-238 (Kabat numbering) and / or positions 230-238 (Chothia numbering) of the hinge are deleted. In an embodiment, one or more of the residues between positions 218- 226 exclusive (EU numbering) are deleted, i.e. not including residue 218 or 226.

[0545] In an embodiment, one, two, three, four or five of the amino acids in positions 5-10 (IMGT hinge numbering), positions 220-225 (EU numbering), positions 233-238 (Kabat numbering) and / or positions 233-238 (Chothia numbering) of the hinge are substituted. Preferably the residues are substituted for residues in the cleavable domain, such residues in the cleavage site and / or linker domain. In an embodiment, the residues in one or more of the following positions are left intact:

[0546] • position 4 (IMGT hinge numbering IgG4), 229 (Kabat numbering) and / or 229 (Chothia numbering); and / or

[0547] • position 8 (IMGT hinge numbering IgG4), 226 (EU numbering), 239 (Kabat numbering) and / or 239 (Chothia numbering); and / or

[0548] • position 9 (IMGT hinge numbering IgG4), 227 (EU numbering), 240 (Kabat numbering) and / or 240 (Chothia numbering); and / or

[0549] • position 10 (IMGT hinge numbering IgG4), 228 (EU numbering), 241 (Kabat numbering) and / or 241 (Chothia numbering); and / or

[0550] • position 11 (IMGT hinge numbering IgG4), 229 (EU numbering), 242 (Kabat numbering) and / or 242 (Chothia numbering); and / or

[0551] • position 12 (IMGT hinge numbering IgG4), 230 (EU numbering), 243 (Kabat numbering) and / or 243 (Chothia numbering).

[0552] In another embodiment, the agent comprises (native) amino acid residues at one or more (e.g. all) of the above positions.

[0553] In an embodiment, the residues in one or more of the following positions are left intact:

[0554] • position 5 (IMGT hinge numbering IgGl), 220 (EU numbering), 233 (Kabat numbering) and / or 233 (Chothia numbering); and / or

[0555] • position 11 (IMGT hinge numbering IgGl), 226 (EU numbering), 239 (Kabat numbering) and / or 239 (Chothia numbering); and / or

[0556] • position 12 (IMGT hinge numbering IgGl), 227 (EU numbering), 240 (Kabat numbering) and / or 240 (Chothia numbering); and / or

[0557] • position 13 (IMGT hinge numbering IgGl), 228 (EU numbering), 241 (Kabat numbering) and / or 241 (Chothia numbering); and / or

[0558] • position 14 (IMGT hinge numbering IgGl), 229 (EU numbering), 242 (Kabat numbering) and / or 242 (Chothia numbering); and / or

[0559] • position 15 (IMGT hinge numbering IgGl), 230 (EU numbering), 243 (Kabat numbering) and / or 243 (Chothia numbering).

[0560] Optionally, position 1.6 (IMGT unique numbering for C-domain CH2), 231 (EU numbering), 244 (Kabat numbering) and / or 244 (Chothia numbering) may be left intact.

[0561] By "intact", we include that the residue at this position is not mutated or changed, e.g. due to the presence of the cleavable domain and / or cleavage site. In another embodiment, the agent comprises (native) amino acid residues at one or more (e.g. all) of the above positions. Middle hinge

[0562] In a particular embodiment, the cleavable domain and / or cleavage site is not located in the middle hinge.

[0563] In other embodiments, the cleavage domain comprises native or intact (as defined elsewhere herein) amino acid residues from the middle hinge region, for example a CPP / SC motif (SEQ ID NOs:79 and 99).

[0564] In IgG antibodies the middle hinge may be located from:

[0565] • positions 226 to 229 (inclusive), as defined by EU numbering;

[0566] • positions 239 to 242 (inclusive), as defined by Kabat numbering; and / or

[0567] • positions 239 to 242 (inclusive), as defined by Chothia numbering.

[0568] For IgGl type antibodies, the middle hinge may be located from:

[0569] • positions 11 to 14 (inclusive), as defined by IMGT hinge numbering;

[0570] • positions 226 to 229 (inclusive), as defined by EU numbering;

[0571] • positions 239 to 242 (inclusive), as defined by Kabat numbering; and / or

[0572] • positions 239 to 242 (inclusive), as defined by Chothia numbering.

[0573] For IgGl type antibodies, the middle hinge may comprise:

[0574] • a cysteine residue at position 11 (IMGT hinge numbering scheme), position 226 (EU numbering scheme), position 239 (Kabat numbering) and / or position 239 (Chothia numbering); and / or

[0575] • a proline residue at position 12 (IMGT hinge numbering scheme), position 221 (EU numbering scheme), position 240 (Kabat numbering) and / or position 240 (Chothia numbering); and / or

[0576] • a proline residue at position 13 (IMGT hinge numbering scheme), position 228 (EU numbering scheme), position 241 (Kabat numbering) and / or position 241 (Chothia numbering); and / or

[0577] • a cysteine residue at position 14 (IMGT hinge numbering scheme), position 229 (EU numbering scheme), position 242 (Kabat numbering) and / or position 242 (Chothia numbering).

[0578] Thus, in one embodiment, the agent (e.g. as part of the cleavable domain) comprises a middle hinge disulphide bond region comprising the sequence CPPC (SEQ ID NO: 79).

[0579] Preferably, position 11 (IMGT hinge numbering scheme), position 226 (EU numbering scheme), position 239 (Kabat numbering scheme) and / or position 239 (Chothia numbering scheme) are not mutated or changed. This position defines the start of the 'middle hinge' region necessary for the linkage of the two heavy chains.

[0580] Preferably, position 12 (IMGT hinge numbering scheme), position 221 (EU numbering scheme), position 240 (Kabat numbering scheme) and / or position 240 (Chothia numbering scheme) are not mutated or changed.

[0581] Preferably, position 13 (IMGT hinge numbering scheme), position 228 (EU numbering scheme), position 241 (Kabat numbering scheme) and / or position 241 (Chothia numbering scheme) are not mutated or changed.

[0582] Preferably, position 14 (IMGT hinge numbering scheme), position 229 (EU numbering scheme), position 242 (Kabat numbering scheme) and / or position 242 (Chothia numbering scheme) are not mutated or changed. This position defines the end of the 'middle hinge' region necessary for the linkage of the two heavy chains.

[0583] For IgG4 type antibodies, the middle hinge may be located from

[0584] • positions 8 to 11 (inclusive), as defined by IMGT hinge numbering;

[0585] • positions 226 to 229 (inclusive), as defined by EU numbering;

[0586] • positions 239 to 242 (inclusive), as defined by Kabat numbering; and / or

[0587] • positions 239 to 242 (inclusive), as defined by Chothia numbering.

[0588] For IgG4 type antibodies, the middle hinge may comprise:

[0589] • a cysteine residue at position 8 (IMGT hinge numbering scheme), position 226 (EU numbering scheme), position 239 (Kabat numbering) and / or position 239 (Chothia numbering); and / or

[0590] • a proline residue at position 9 (IMGT hinge numbering scheme), position 221 (EU numbering scheme), position 240 (Kabat numbering) and / or position 240 (Chothia numbering); and / or

[0591] • a serine residue at position 10 (IMGT hinge numbering scheme), position 228 (EU numbering scheme), position 241 (Kabat numbering) and / or position 241 (Chothia numbering); and / or

[0592] • a cysteine residue at position 11 (IMGT hinge numbering scheme), position 229 (EU numbering scheme), position 242 (Kabat numbering) and / or position 242 (Chothia numbering).

[0593] Thus in one embodiment, the agent (e.g. as part of the cleavable domain) comprises a middle hinge disulphide bond region comprising the sequence CPSC (SEQ ID NO: 149). Preferably, position 8 (IMGT hinge numbering scheme), position 226 (EU numbering scheme), position 239 (Kabat numbering scheme) and / or position 239 (Chothia numbering scheme) are not mutated or changed. This position defines the start of the 'middle hinge' region necessary for the linkage of the two heavy chains.

[0594] Preferably, position 9 (IMGT hinge numbering scheme), position 221 (EU numbering scheme), position 240 (Kabat numbering scheme) and / or position 240 (Chothia numbering scheme) are not mutated or changed.

[0595] Preferably, position 10 (IMGT hinge numbering scheme), position 228 (EU numbering scheme), position 241 (Kabat numbering scheme) and / or position 241 (Chothia numbering scheme) are not mutated or changed.

[0596] Preferably, position 11 (IMGT hinge numbering scheme), position 229 (EU numbering scheme), position 242 (Kabat numbering scheme) and / or position 242 (Chothia numbering scheme) are not mutated or changed. This position defines the end of the 'middle hinge' region necessary for the linkage of the two heavy chains.

[0597] Cleavable domain in lower hinge

[0598] In some embodiments, the cleavable domain and / or the cleavage site may be located in the lower hinge. In some embodiments, the cleavable domain and / or the cleavage site may be located in the lower hinge / CH2 domain. This may be beneficial as it allows for the generation of F(ab')2 fragments following cleavage.

[0599] / . IgGl type antibodies or Fc regions derived therefrom

[0600] The cleavable domain and / or the cleavage site may be located in a hinge region of an IgGl antibody or an antibody derived therefrom, optionally the lower hinge region. The cleavable domain and / or cleavage site may be introduced into the hinge region by insertion and / or substitution.

[0601] In some embodiments, N-terminal proline of the lower hinge is left intact. By "intact", we include that the residue at this position is not mutated or changed, e.g. due to the presence of the cleavable domain and / or cleavage site. In another embodiment, the agent comprises an N-terminal proline of the lower hinge. For example, the N-terminal proline of the lower hinge is not deleted and / or substituted. The cleavable domain and / or cleavage site is located before or after the N-terminal cysteine of the lower hinge. Preferably, the cleavable domain and / or cleavage site is located after the N-terminal cysteine of the CHI domain (i.e. after the C terminus of the proline). By "N-terminal proline of the lower hinge", we include the proline at position 15 (IMGT IgGl hinge numbering), position 230 (EU numbering), position 243 (Kabat numbering) and / or position 243 (Chothia numbering) in an IgGl antibody or the cysteine at the corresponding position on other antibody types. In one embodiment, the agent comprises a proline at position 15 (IMGT hinge numbering), position 230 (EU numbering), position 243 (Kabat numbering) and / or position 243 (Chothia numbering) in an IgGl antibody or the cysteine at the corresponding position on other antibody formats.

[0602] In an embodiment, the cleavable domain and / or cleavage site is located after:

[0603] • position 15 as defined by IMGT hinge numbering;

[0604] • position 230 as defined by EU numbering;

[0605] • position 243 as defined by Kabat numbering; and / or

[0606] • position 243 as defined by Chothia numbering.

[0607] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0608] • position 5 as defined by IMGT unique numbering for C-domain;

[0609] • position 241 as defined by EU numbering;

[0610] • position 255 as defined by Kabat numbering; and / or

[0611] • position 255 as defined by Chothia numbering.

[0612] Additionally or alternatively, the cleavable domain and / or cleavage site ends at:

[0613] • position 4 as defined by IMGT unique numbering for C-domain;

[0614] • position 240 as defined by EU numbering;

[0615] • position 253 as defined by Kabat numbering; and / or

[0616] • position 253 as defined by Chothia numbering.

[0617] The cleavable domain and / or cleavage site may be:

[0618] • located after position 15 (IMGT hinge numbering) and is located before position 5 (IMGT unique numbering for C-domain);

[0619] • located after position 230 and is located before position 241, as defined by EU numbering;

[0620] • located after position 243 and is located before position 255, as defined by Kabat numbering; and / or

[0621] • located after position 243 and is located before position 255, as defined by Chothia numbering.

[0622] The cleavable domain and / or cleavage site may be:

[0623] • located after position 15 (IMGT hinge numbering) and ends by position 4 (IMGT unique numbering for C-domain);

[0624] • located after position 229 and ends by position 240, as defined by EU numbering; • located after position 242 and ends by position 243, as defined by Kabat numbering; and / or

[0625] • located after position 242 and ends by position 243, as defined by Chothia numbering.

[0626] In a preferred embodiment, the cleavable domain and / or cleavage site is located after:

[0627] • position 1.6 as defined by IMGT unique numbering for C-domain;

[0628] • position 231 as defined by EU numbering;

[0629] • position 244 as defined by Kabat numbering; and / or

[0630] • position 244 as defined by Chothia numbering.

[0631] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0632] • position 1.5 as defined by IMGT unique numbering for C-domain;

[0633] • position 232 as defined by EU numbering;

[0634] • position 245 as defined by Kabat numbering; and / or

[0635] • position 245 as defined by Chothia numbering.

[0636] Additionally or alternatively, the cleavable domain and / or cleavage site is ends at:

[0637] • position 1.5 as defined by IMGT unique numbering for C-domain;

[0638] • position 232 as defined by EU numbering;

[0639] • position 245 as defined by Kabat numbering; and / or

[0640] • position 245 as defined by Chothia numbering.

[0641] The cleavable domain and / or cleavage site may be:

[0642] • located after position 1.6 and is located before position 1.5, as defined by IMGT unique numbering for C-domain;

[0643] • located after position 231 and is located before position 232, as defined by EU numbering;

[0644] • located after position 244 and is located before position 245, as defined by Kabat numbering; and / or

[0645] • located after position 244 and is located before position 245, as defined by Chothia numbering.

[0646] The cleavable domain and / or cleavage site may be:

[0647] • located after position 1.6 and ends by position 1.5, as defined by IMGT unique numbering for C-domain;

[0648] • located after position 231 and ends by position 232, as defined by EU numbering;

[0649] • located after position 244 and ends by position 245, as defined by Kabat numbering; and / or located after position 244 and ends by position 245, as defined by Chothia numbering.

[0650] The cleavable domain and / or cleavage site may be engineered into the hinge as an insertion between:

[0651] • positions 1.6 and 1.5, as defined by IMGT unique numbering for C-domain;

[0652] • positions 231 and 232, as defined by EU numbering;

[0653] • positions 244 and 245, as defined by Kabat numbering; and / or

[0654] • positions 244 and 245, as defined by Chothia numbering.

[0655] In an embodiment, the residues in the hinge region are substituted by residues from the cleavable domain (e.g. the cleavage site and / or linker domain(s)) at the following positions:

[0656] • positions 1.6 to 4, as defined by IMGT unique numbering for C-domain;

[0657] • positions 233 to 240, as defined by EU numbering;

[0658] • positions 246 to 253, as defined by Kabat numbering; and / or

[0659] • positions 246 to 253, as defined by Chothia numbering.

[0660] In an embodiment, the residues in one or more of the following positions are left intact: position 11 (IMGT hinge numbering IgGl), 226 (EU numbering), 239 (Kabat numbering) and / or 239 (Chothia numbering); and / or position 12 (IMGT hinge numbering IgGl), 227 (EU numbering), 240 (Kabat numbering) and / or 240 (Chothia numbering); and / or position 13 (IMGT hinge numbering IgGl), 228 (EU numbering), 241 (Kabat numbering) and / or 241 (Chothia numbering); and / or position 14 (IMGT hinge numbering IgGl), 229 (EU numbering), 242 (Kabat numbering) and / or 242 (Chothia numbering); and / or position 15 (IMGT hinge numbering IgGl), 230 (EU numbering), 243 (Kabat numbering) and / or 243 (Chothia numbering); and / or position 5 (IMGT unique numbering for C-domain CH2), 241 (EU numbering), 255 (Kabat numbering) and / or 255 (Chothia numbering); and / or position 6 (IMGT unique numbering for C-domain CH2), 242 (EU numbering), 256

[0661] (Kabat numbering) and / or 256 (Chothia numbering); and / or position 4 (IMGT unique numbering for C-domain CH2), 243 (EU numbering), 257 (Kabat numbering) and / or 257 (Chothia numbering).

[0662] By "intact", we include that the residue at this position is not mutated or changed, e.g. due to the presence of the cleavable domain and / or cleavage site. In another embodiment, the agent comprises (native) amino acid residues at one or more (e.g. all of) the above positions.

[0663] / / . IgG4 type antibodies or Fc regions derived therefrom The cleavable domain and / or the cleavage site may be located in a hinge region of an IgG4 antibody or an antibody derived therefrom, optionally the lower hinge region. The cleavable domain and / or cleavage site may be introduced into the hinge region by insertion and / or substitution.

[0664] In some embodiments, N-terminal proline of the lower hinge is left intact. By "intact", we include that the residue at this position is not mutated or changed, e.g. due to the presence of the cleavable domain and / or cleavage site. In another embodiment, the agent comprises an N-terminal proline of the lower hinge. For example, the N-terminal proline of the lower hinge is not deleted and / or substituted. The cleavable domain and / or cleavage site is located before or after the N-terminal cysteine of the lower hinge. Preferably, the cleavable domain and / or cleavage site is located after the N-terminal cysteine of the CHI domain (i.e. after the C terminus of the proline). By "N-terminal proline of the lower hinge", we include the proline at position 12 (IMGT IgG4 hinge numbering), position 230 (EU numbering), position 243 (Kabat numbering) and / or position 243 (Chothia numbering) in an IgGl antibody or the cysteine at the corresponding position on other antibody types. In one embodiment, the agent comprises a proline at position 15 (IMGT hinge numbering), position 230 (EU numbering), position 243 (Kabat numbering) and / or position 243 (Chothia numbering) in an IgGl antibody or the cysteine at the corresponding position on other antibody formats.

[0665] In an embodiment, the cleavable domain and / or cleavage site is located after:

[0666] • position 12 as defined by IMGT IgG4 hinge numbering;

[0667] • position 230 as defined by EU numbering;

[0668] • position 243 as defined by Kabat numbering; and / or

[0669] • position 243 as defined by Chothia numbering.

[0670] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0671] • position 5 as defined by IMGT unique numbering for C-domain;

[0672] • position 241 as defined by EU numbering;

[0673] • position 255 as defined by Kabat numbering; and / or

[0674] • position 255 as defined by Chothia numbering.

[0675] Additionally or alternatively, the cleavable domain and / or cleavage site ends at:

[0676] • position 4 as defined by IMGT unique numbering for C-domain;

[0677] • position 240 as defined by EU numbering;

[0678] • position 253 as defined by Kabat numbering; and / or

[0679] • position 253 as defined by Chothia numbering.

[0680] The cleavable domain and / or cleavage site may be: • located after position 12 (IMGT IgG4 hinge numbering) and is located before position 5 (IMGT unique numbering for C-domain);

[0681] • located after position 230 and is located before position 241, as defined by EU numbering;

[0682] • located after position 243 and is located before position 255, as defined by Kabat numbering; and / or

[0683] • located after position 243 and is located before position 255, as defined by Chothia numbering.

[0684] The cleavable domain and / or cleavage site may be:

[0685] • located after position 12 (IMGT IgG4 hinge numbering) and ends by position 4 (IMGT unique numbering for C-domain);

[0686] • located after position 229 and ends by position 240, as defined by EU numbering;

[0687] • located after position 242 and ends by position 243, as defined by Kabat numbering; and / or

[0688] • located after position 242 and ends by position 243, as defined by Chothia numbering.

[0689] In a preferred embodiment, the cleavable domain and / or cleavage site is located after:

[0690] • position 1.6 as defined by IMGT unique numbering for C-domain;

[0691] • position 231 as defined by EU numbering;

[0692] • position 244 as defined by Kabat numbering; and / or

[0693] • position 244 as defined by Chothia numbering.

[0694] Additionally or alternatively, the cleavable domain and / or cleavage site is located before:

[0695] • position 1.5 as defined by IMGT unique numbering for C-domain;

[0696] • position 232 as defined by EU numbering;

[0697] • position 245 as defined by Kabat numbering; and / or

[0698] • position 245 as defined by Chothia numbering.

[0699] Additionally or alternatively, the cleavable domain and / or cleavage site is ends at:

[0700] • position 1.5 as defined by IMGT unique numbering for C-domain;

[0701] • position 232 as defined by EU numbering;

[0702] • position 245 as defined by Kabat numbering; and / or

[0703] • position 245 as defined by Chothia numbering.

[0704] The cleavable domain and / or cleavage site may be:

[0705] • located after position 1.6 and is located before position 1.5, as defined by IMGT unique numbering for C-domain; • located after position 231 and is located before position 232, as defined by EU numbering;

[0706] • located after position 244 and is located before position 245, as defined by Kabat numbering; and / or

[0707] • located after position 244 and is located before position 245, as defined by Chothia numbering.

[0708] The cleavable domain and / or cleavage site may be:

[0709] • located after position 1.6 and ends by position 1.5, as defined by IMGT unique numbering for C-domain;

[0710] • located after position 231 and ends by position 232, as defined by EU numbering;

[0711] • located after position 244 and ends by position 245, as defined by Kabat numbering; and / or

[0712] • located after position 244 and ends by position 245, as defined by Chothia numbering.

[0713] The cleavable domain and / or cleavage site may be engineered into the hinge as an insertion between:

[0714] • positions 1.6 and 1.5, as defined by IMGT unique numbering for C-domain;

[0715] • positions 231 and 232, as defined by EU numbering;

[0716] • positions 244 and 245, as defined by Kabat numbering; and / or

[0717] • positions 244 and 245, as defined by Chothia numbering.

[0718] In an embodiment, the residues in the hinge region are substituted by residues from the cleavable domain (e.g. the cleavage site and / or linker domain(s)) at the following positions:

[0719] • positions 1.6 to 4, as defined by IMGT unique numbering for C-domain;

[0720] • positions 233 to 240, as defined by EU numbering;

[0721] • positions 246 to 253, as defined by Kabat numbering; and / or

[0722] • positions 246 to 253, as defined by Chothia numbering.

[0723] In an embodiment, the residues in one or more of the following positions are left intact:

[0724] • position 8 (IMGT hinge numbering IgG4), 226 (EU numbering), 239 (Kabat numbering) and / or 239 (Chothia numbering); and / or

[0725] • position 9 (IMGT hinge numbering IgG4), 227 (EU numbering), 240 (Kabat numbering) and / or 240 (Chothia numbering); and / or

[0726] • position 10 (IMGT hinge numbering IgG4), 228 (EU numbering), 241 (Kabat numbering) and / or 241 (Chothia numbering); and / or

[0727] • position 11 (IMGT hinge numbering IgG4), 229 (EU numbering), 242 (Kabat numbering) and / or 242 (Chothia numbering); and / or • position 12 (IMGT hinge numbering IgG4), 230 (EU numbering), 243 (Kabat numbering) and / or 243 (Chothia numbering); and / or

[0728] • position 5 (IMGT unique numbering for C-domain CH2), 241 (EU numbering), 255 (Kabat numbering) and / or 255 (Chothia numbering); and / or

[0729] • position 6 (IMGT unique numbering for C-domain CH2), 242 (EU numbering), 256 (Kabat numbering) and / or 256 (Chothia numbering); and / or

[0730] • position 4 (IMGT unique numbering for C-domain CH2), 243 (EU numbering), 257 (Kabat numbering) and / or 257 (Chothia numbering).

[0731] In some embodiments, the cleavable domain comprises a sequence that is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to sequences selected from the group consisting of:

[0732] Cleavable domain of Engineered trastuzumab design lx (SEQ ID NO: 30)

[0733] GSLSGRSDNHDKTHTCPPCPAP

[0734] Cleavable domain of Engineered trastuzumab design 1 (SEQ ID NO: 146) GSLSGRSDNHASDKTHTCPPCPAP

[0735] Cleavable domain of Engineered trastuzumab design 2x (SEQ ID NO: 31) LSGRSDNHDKTHTCPPCPAP

[0736] Cleavable domain of Engineered trastuzumab design 2 (SEQ ID NO: 147) LSGRSDNHASDKTHTCPPCPAP

[0737] Cleavable domain of Engineered trastuzumab design 3x (SEQ ID NO: 32) GSLSGRSDNHGSDKTHTCPPCPAP

[0738] Cleavable domain of Engineered trastuzumab design 3 (SEQ ID NO: 148)

[0739] GSLSGRSDNHGSASDKTHTCPPCPAP

[0740] Cleavable domain of Engineered trastuzumab design 4 and 4x (SEQ ID NO: 33) LSGRSDNHCPPCPAP

[0741] Cleavable domain of Engineered trastuzumab design 5 and 5x (SEQ ID NO: 34)

[0742] LSGRSDNHTC P PC PAP

[0743] Cleavable domain of Engineered trastuzumab designs 6x and 7x (SEQ ID NO: 35) DKTHTCPPCPAPLSGRSDNH Cleavable domain of Engineered trastuzumab designs 6 and 7 (SEQ ID NO: 152) DKTHTCPPCPALSGRSDNH

[0744] Cleavable domain of Engineered trastuzumab design 9 (SEQ ID NO: 36)

[0745] GSPRAEALKGGGSASDKTHTCPPCPAP

[0746] Cleavable domain of Engineered designs 12, 13, 15 and 16 (SEQ ID NO: 37) GSLSGRSDNHGSASDKTHTCPPCPAP

[0747] Cleavable domain of Engineered pembrolizumab design 14a (SEQ ID NO: 38) GSLSGRSDNHGSGPPCPPCPAP

[0748] Cleavable domain of Engineered pembrolizumab design 14b (SEQ ID NO: 39) GPPGSLSGRSDNHGSCPPCPAP

[0749] Cleavable domain of Engineered pembrolizumab design 14c (SEQ ID NO: 102)

[0750] GSLSGRSDNHGSNVDHKPSNTKVDKRVESKYGPPCPPCPAP

[0751] Cleavable domain of Engineered trastuzumab design 17 (SEQ ID NO: 40) LSGRSDNHGSASDKTHTCPPCPAP

[0752] Cleavable domain of Engineered trastuzumab design 18 (SEQ ID NO: 41)

[0753] GGGSGGGSLSGRSDNHGSASDKTHTCPPCPAP

[0754] Cleavable domain of PEGylated trastuzumab design 20 (SEQ ID NO: 103) GGGSGGGSLSGRSDNHGGGSHHHHHHHH

[0755] In one embodiment, the protease that acts on the cleavage site is expressed by and / or accumulates in the vicinity of unwanted cells or a tumour. Preferably, the protease is a tumourspecific protease. Thus, when the unwanted cells are tumour cells, the cleavage site may be cleavable selectively by proteases that are found in the vicinity of the tumour cells.

[0756] Specificity of the agent may be increased by virtue of the cleavage site only being cleaved in the vicinity of the unwanted cells. For example, tumour cells secrete proteases that are required by tumours for invasion of local tissues and metastasis, and so by including a tumourspecific protease cleavage site in the agent, the specificity of the agent for the tumour may be increased.

[0757] "Vicinity" or "tumour microenvironment" herein refers to the area at and / or near to the surface of the cells, such as the environment that immediately surrounds the cells e.g. blood vessels, immune cells, fibroblasts, signalling molecules and / or extracellular matrix (ECM), blood, lymph, and other body fluids. In one embodiment, the vicinity starts at the tumour site and extends at least 10 micrometres (pm), at least 20 pm, at least 30 pm, at least 40 pm, at least 50 pm, at least 60 pm, at least 70 pm, at least 80 pm, at least 90 pm, at least 100 pm, at least 120 pm, at least 140 pm, at least 160 pm, at least 180 pm, at least 200 pm away from the tumour.

[0758] In some embodiments, the concentration of enzymes may be low in some tissue where the agents of the invention are not cleaved and do not penetrate deeper, for example in nondisease tissue. In some embodiments, the enzymes are not active or are significantly less active in healthy (e.g. non-diseased) tissue or tissues not intended for therapy.

[0759] It is also appreciated that since the cleavage site in the agent may confer specificity on where the therapeutic domain is released, binding of therapeutic domain to non-disease cells, in the vicinity of which the cleavage site is not cleaved, may also be tolerated. This may result in reduced (i.e. fewer and / or less severe) side effects, which may lead to increased patient compliance and / or uptake of these agents, e.g. as compared to other cancer therapies.

[0760] The proteases may include any of a cysteine protease (including the Cathepsin family B, L, S etc), an aspartyl or aspartic protease (including Cathepsin D and E, or Napsin A) and a serine protease (including Cathepsin A and G, Thrombin, Plasmin, uPA, tissue Plasminogen Activator (tPA), matriptase, MT-SP1, fibroblast activation protein (FAP)).

[0761] The protease may be a metalloproteinase (also known as a metallopeptidase or metalloproteinase) (MMP1-28) including both membrane-bound (MMP14-17 and MMP24-25) and secreted forms (MMP1-13 and MMP18-23 and MMP26-28). The protease may belong to the A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin, or Metalloproteinase with Thrombospondin Motifs (ADAMTS) families of proteases. Other examples include CD10 (CALLA), prostate specific antigen (PSA) and coagulation factors. It is appreciated that the proteases may or may not be membrane-bound. The proteases may include any of them in combination thereof.

[0762] In order to treat a particular tumour type, the skilled person will typically select a protease cleavage site that is selectively cleaved by a protease known to be highly expressed in that tumour type. Tumour specific proteases are well characterized in the literature as targets for drug discovery (small molecules, peptides, antibodies) and also as biomarkers of severity of oncological indications. Tumour specific proteases are usually surface bound proteases that are membrane bound, and are typically inactive. They may be cleaved off in response to disease (although the exact mechanisms are unclear) and become activated and are associated with tumour suppression and metastasis. Different oncological indications have prominent proteases that are associated with disease. For example, to treat breast cancer, it is preferred to use a protease cleavage site cleavable by any of uPA, Napsin A, tPA, legumain, matriptase, matriptase 2, Cathepsin K, Cathepsin O, MMP1, MMP2, MMP3, MMP11, MMP12, MMP17, ADAM9, ADAM10, ADAM12, ADAM15, ADAM17, ADAM28 or ADAMTS15, and so on.

[0763] In a preferred embodiment, the protease is uPA and the cleavable domain comprises a cleavage site that is specifically cleaved by uPA (e.g. LSGRSDNH, SEQ ID NO: 77). In any of the aspects, embodiments or disclosures, the cleavable domain may comprise a uPA protease cleavage site comprising or consisting of the sequence: LSGRSDNH (SEQ ID NO: 77).

[0764] In one embodiment the cleavage site comprises or consists of a variant sequence of SEQ ID NO: 77, for example a sequence with 1, 2, or 3 amino acid insertions, deletions or substitutions relative to SEQ ID NO: 77, but wherein the cleavage site activity is retained - i.e. the variant sequence is still capable of being specifically cleaved by uPA. uPA has a strong association with cancers, including triple negative breast cancer, prostate cancer and gastric cancer. We anticipate screening both the serum and tumour biopsies from patients in the future to determine the expression of both the antigen of interest, and protease of interest to deliver the best treatment. These methods are described elsewhere herein.

[0765] In a preferred embodiment, the protease is ADAM 10 and the cleavable domain comprises a cleavage site that is specifically cleaved by ADAM10 (e.g. PRAEALKGG (SEQ ID NO: 89)). In any of the aspects, embodiments or disclosures, the cleavable domain may comprise an ADAM10 protease cleavage site comprising or consisting of the sequence: PRAEALKGG (SEQ ID NO: 89).

[0766] In one embodiment the cleavage site comprises or consists of a variant sequence of SEQ ID NO: 89, for example a sequence with 1, 2, or 3 amino acid insertions, deletions or substitutions relative to SEQ ID NO: 89, but wherein the cleavage site activity is retained - i.e. the variant sequence is still capable of being specifically cleaved by ADAM 10.

[0767] In some embodiments, the cleavable domain comprises one or more linker domains present between the therapeutic domain and the stabilisation domain.

[0768] Such linker domains can allow for greater accessibility of the enzyme and be selected from the group consisting of amino acid sequences represented by (GmSn)x or (GGNGT)X(SEQ ID NO: 42) or (YGNGT)x (SEQ ID NO: 43) wherein m and n are each independently selected from the group consisting of integers from 1 to 8 (e.g., 1, 2, 3, 4, 5, 6, 7 or 8), and x is independently selected from the group consisting of integers from 1 to 20 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20). In some specific embodiments, the linker has an amino acid sequence represented by (G4S)x (SEQ ID NO: 44), wherein x is independently selected from the group consisting of integers from 1 to 6 (e.g., x is 4 or 5) such as (G4S)2 (SEQ ID NO: 45). The linker domain may also be another linker having similar flexibility and length to the amino acid sequence represented by (GmSn)x or (GGNGT)X(SEQ ID NO: 42) or (YGNGT)x (SEQ ID NO: 43) described above; the linker domain may also be selected from the group consisting of GPPGS (SEQ ID NO: 46), GSGPP (SEQ ID NO: 47), AKTTPKLEEGEFSEAR (SEQ ID NO: 48), AKTTPKLEEGEFSEARV (SEQ ID NO: 49), AKTTPKLGG (SEQ ID NO: 50), SAKTTPKLGG (SEQ ID NO: 51), AKTTPKLEEGEFSEARV (SEQ ID NO: 52), SAKTTP (SEQ ID NO: 53), SAKTTPKLGG (SEQ ID NO: 54), RADAAP (SEQ ID NO: 55), RADAAPTVS (SEQ ID NO: 56), RADAAAAGGPGS (SEQ ID NO: 57), RADAAAA(G4S)4(SEQ ID NO: 58), SAKTTP (SEQ ID NO: 59), SAKTTPKLGG (SEQ ID NO: 60), SAKTTPKLEEGEFSEARV (SEQ ID NO: 61), ADAAP (SEQ ID NO: 62), ADAAPTVSIFPP (SEQ ID NO: 63), TVAAP (SEQ ID NO: 64), TVAAPSVFIFPP (SEQ ID NO: 65), QPKAAP (SEQ ID NO: 66), QPKAAPSVTLFPP (SEQ ID NO: 67), AKTTPP (SEQ ID NO: 68), AKTTPPSVTPLAP (SEQ ID NO: 69), AKTTAP (SEQ ID NO: 70), AKTTAPSVYPLAP (SEQ ID NO: 71), ASTKGP (SEQ ID NO: 72), ASTKGPSVFPLAP (SEQ ID NO: 73), GENKVEYAPALMALS (SEQ ID NO: 74), GPAKELTPLKEAKVS (SEQ ID NO: 75) and GHEAAAVMQVQYPAS (SEQ ID NO: 76). Such linker domains may comprise (GGGS)n, e.g. the linker domain may comprise or consist of one of the following linker domains: GGGSGGGS (SEQ ID NO: 141); GGGS (SEQ ID NO: 142). The linker may comprise the sequence of any of the linker sequences described herein with AS on the N or C-terminal end, preferably the N-terminal end.

[0769] In some embodiments, the linker domain is from 1 to 3, from 2 to 4, from 2 to 6, from 2 to 8, from 2 to 10, from 2 to 12, from 2 to 14, from 2 to 16, from 2 to 18, from 2 to 20, from 2 to 30 or from 2 to 40 amino acids in length. Preferably, the linker domain is between 2 and 16 amino acids in length. One or more linker domain(s) may be located at the C-terminus of the cleavage site and / or the N-terminus of the cleavage site (e.g. with 1, 2 or 3 residues of the ends of the cleavage site). Where the linker domain(s) are not immediately adjacent to the cleavage site, one or more (native or intact) amino acid residues from the parental (e.g. original) molecule may be present.

[0770] In an embodiment, the linker domain comprises or consists of neutral amino acids. Preferably, at least 70%, 80%, 90%, 95% or 100% of the amino acids in the linker domain are neutral amino acids.

[0771] By "neutral amino acids" we include amino acids that possess an equal number of amino and carboxylic acid groups. Examples of neutral amino acids include glycine, alanine, leucine, isoleucine, valine, phenylalanine, proline, methionine, serine, threonine, tyrosine, glutamine, asparagine and tryptophan.

[0772] In an embodiment, the linker domain comprises or consists of S, G, A and / or T. Preferably, at least 70%, 80%, 90%, 95% or 100% of the amino acids in the linker domain are S, G, A or T. In an embodiment, the linker domain comprises or consists of a G / S rich, e.g. the linker domain may comprise at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and / or at least 95% G or S residues. Increased levels of G / S in linker domain sequences may be beneficial as they provide greater accessibility to the cleavage site.

[0773] In an embodiment, the linker domain comprises spacer sequences comprising A, G and / or T. For example, the linker domain may comprise at least at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and / or at least 95% A / G / T residues.

[0774] In an embodiment the agent comprises a His-tag (HHHHHH, SEQ ID NO: 143).

[0775] In some embodiments, the cleavable domain overlaps with the therapeutic domain and / or the stabilisation domain. In a particular embodiment, the cleavable domain does not overlap with the therapeutic domain and / or the stabilisation domain.

[0776] In one embodiment, the cleavable domain may be an additional sequence (which adds further amino acid to the biologic). In another embodiment, the cleavable domain is generated by creating one or more mutations (e.g. addition, substitution or deletion) to the endogenous (native, original) sequence (for example, which does not add any further amino acids to the biologic). In yet another embodiment, it could be a combination of the addition of a sequence and mutagenesis.

[0777] In some embodiments, the cleavable domain overlaps with at least 2%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 99%, at least 100% with the therapeutic domain and / or stabilisation domain.

[0778] Typically, small cleavage sites or small cleavable domains (including any linkers if present) are favourable because the risk of immunogenicity (to foreign / non-host sequences) may be lower.

[0779] In some embodiments, the at least one cleavage site and / or the cleavable domain is 4 to 20 amino acids in length. For example, the at least one cleavage site and / or the cleavable domain has a length of 4 to 6 amino acids, 4 to 8 amino acids, 4 to 10 amino acids, 4 to 12 amino acids, 4 to 14 amino acids, 4 to 16 amino acids, 4 to 18 amino acids, 6 to 8 amino acids, 6 to 10 amino acids, 6 to 12 amino acids, 6 to 14 amino acids, 6 to 16 amino acids, 6 to 18 amino acids, 6 to 20 amino acids, 8 to 10 amino acids, 8 to 12 amino acids, 8 to 14 amino acids, 8 to 16 amino acids, 8 to 18 amino acids, 8 to 20 amino acids, 10 to 12 amino acids, 10 to 14 amino acids, 10 to 16 amino acids, 10 to 18 amino acids, 10 to 20 amino acids, 12 to 14 amino acids, 12 to 16 amino acids, 12 to 18 amino acids, 12 to 20 amino acids, 14 to 16 amino acids, 14 to 18 amino acids, 14 to 20 amino acids, 16 to 18 amino acids, 16 to 20 amino acids, 18 to 20 amino acids. Suitably the cleavage site and / or the cleavable domain is in the lower hinge, preferably when the length is one of the aforementioned ranges that have up to 16 amino acids. Suitably the cleavage site and / or the cleavable domain is in the upper hinge, preferably when the length is one of the aforementioned ranges that have up to 20 amino acids.

[0780] In a further embodiment, the agent is a protein or polypeptide, the stabilisation or the therapeutic domain comprises a Fc region and a hinge region and the cleavable domain is located: i. N-terminal to the hinge region and after the therapeutic domain; or ii. C-terminal to the hinge region and before the CH2 domain of the Fc region.

[0781] In some embodiments, the uncleaved agent has a structural arrangement from N-terminus to C-terminus as follows: i. Therapeutic domain-cleavable domain-Hinge region-Fc region; or ii. Therapeutic domain-hinge region-cleavable domain-Fc region.

[0782] The cleavable domain may or may not occur either before or after the hinge region. This difference in location will give two separate moieties that may yield a monovalent or multivalent (such a bivalent) binding protein. The engineering design will depend on the preferred mode of action for the therapeutic.

[0783] In some embodiments, the cleavable domain is not cleavable by a tumour-specific protease from the list consisting of Gelatinase A (MMP-2), Stromelysin 1 (MMP-3), Matrilysin (MMP-7), Gelatinase B (MMP-9), Macrophage metalloelastase (MMP-12), Collagenase-3 (MMP-13), Cathepsin G9). In another embodiment, the cleavable domain is not cleavable by the protease Capsase-3 or by the PreScission protease. By "PreScission protease", we include the meaning of a fusion of GST and a human rhinovirus protease.

[0784] In a preferred embodiment, the therapeutic domain is a Fab, the cleavable domain comprises a uPA cleavage site, and the stabilisation domain is an Fc region.

[0785] In a preferred embodiment, the therapeutic domain is a Fab, the cleavable domain comprises a ADAM10 cleavage site, and the stabilisation domain is an Fc region.

[0786] In another preferred embodiment, the therapeutic domain is a VHH, the cleavable domain comprises a uPA cleavage site, and the stabilisation domain is an Fc region.

[0787] In a preferred embodiment, the therapeutic domain is a ScFv, the cleavable domain comprises a uPA cleavage site, and the stabilisation domain is an Fc region. In a preferred embodiment, the therapeutic domain is a scFv, the cleavable domain comprises a uPA cleavage site and the stabilisation domain comprises PEGylated domain. The scFv may be a Trastuzumab scFV. The scFv may comprise SEQ ID NO: 25 and / or SEQ ID NO: 26. The PEGylated domain may be attached to the cleavage domain via a HIS-tag. The agent may be as described in Example 10.

[0788] In an embodiment, the agent does not comprise or consist of the amino acid sequence of Dlx, D2x, D3x, D4x, D5x, D6x and / or D7x. In an embodiment, the agent does not comprise or consist of the amino acid sequence of Dlx, D2x, D3x, D4x, D5x, D6x and / or D7x and / or a sequence having at least 97%, 98% or 99% identity thereto

[0789] In an embodiment, the agent does not comprise or consist of a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 92, 97, 98, 105, 107, 109, 111, 113, 115, 117 and / or 120, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0790] In a preferred embodiment, the agent does not comprise or consist of a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 and / or 16 and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In a preferred embodiment, the agent does not comprise or consist of a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16.

[0791] In a preferred embodiment, the agent does not comprise or consist of a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 92, 97 and 98, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In a preferred embodiment, the agent does not comprise or consist of a sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 92, 97 and 98.

[0792] In a preferred embodiment, the therapeutic domain does not comprise or consist of a sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 99 and / or 100, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In a preferred embodiment, the therapeutic domain does not comprise or consist of a sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 99 and / or 100.

[0793] In a preferred embodiment, the therapeutic domain does not comprise or consist of a sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24 and 25, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In a preferred embodiment, the therapeutic domain does not comprise or consist of a sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24 and / or 25. In an embodiment, the stabilisation domain does not comprise or consist of a sequence of SEQ ID NO: 27, 28, 29 and / or 101, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the stabilisation domain does not comprise or consist of a sequence of SEQ ID NO: 27, 28, 29 and / or 101.

[0794] In an embodiment, the stabilisation domain does not comprise or consist of a sequence of SEQ ID NO: 27, 28 and / or 29, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the stabilisation domain does not comprise or consist of a sequence of SEQ ID NO: 27, 28 and / or 29.

[0795] In an embodiment, the agent does not comprise:

[0796] (a) a stabilisation domain comprising or consisting of a sequence of SEQ ID NO: 27, 28 and / or 29, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; and

[0797] (b) a therapeutic domain comprising or consisting of a sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 99 and / or 100, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto; or

[0798] (c) a therapeutic domain comprising or consisting of a sequence of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24 and 25, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto.

[0799] In an embodiment, the cleavable domain does not comprise or consist of a sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 102, 103, 146, 147, 152 and / or 148, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the cleavable domain does not comprise or consist of a sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 102, 103, 146, 147, 152 and / or 148.

[0800] In an embodiment, the cleavable domain does not comprise or consist of a sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 and / or 41, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the cleavable domain does not comprise or consist of a sequence of SEQ ID NO: 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 or 41.

[0801] In an embodiment, the agent does not comprise a linker domain comprising a sequence of SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 141 and / or 142, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the agent does not comprise a linker domain comprising a sequence of SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 141 and / or 142.

[0802] In an embodiment, the agent does not comprise a linker domain comprising a sequence of SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63,

[0803] 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and / or 76 and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the agent does not comprise a linker domain comprising a sequence of SEQ ID NOs: 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,

[0804] 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 and / or 76

[0805] In an embodiment, the agent does not comprise a cleavable domain and / or cleavage site comprising a sequence of SEQ ID NO: 77 or 89.

[0806] In an embodiment, the agent is not encoded by a sequence comprising any one of SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88, 91, 104, 106, 108, 110, 112, 114, 116, 118, 119, 121 and / or 125 and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the agent is not encoded by a sequence comprising any one of SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88, 91, 104, 106, 108, 110, 112, 114, 116, 118, 119, 121 and / or 125.

[0807] In an embodiment, the agent is not encoded by a sequence comprising any one of SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88 and / or 91, and / or a sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity thereto. In an embodiment, the agent is not encoded by a sequence comprising any one of SEQ ID NOs: 82, 83, 84, 85, 86, 87, 88 and / or 91.

[0808] In an embodiment, the agent does not comprise or consist of a sequence of any one of SEQ ID NOs: 1-81, 89, 90, 92-103, 105, 107, 109, 111, 113, 115, 117 and / or 120.

[0809] In an embodiment, the agent does not comprise or consist of a sequence of any one of SEQ ID NOs: 1-81, 89, 90 and / or 92.

[0810] Light chain sequences

[0811] > Trastuzumab parental antibody light chain sequence: SEQ ID NO: 158

[0812] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTD FTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG

[0813] EC

[0814] >Pembrolizumab light chain protein sequence (LC004) - SEQ ID NO: 172

[0815] EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGS GTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFY PREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSF NRG EC

[0816] >M5A light chain protein sequence (LC005) - SEQ ID NO: 173

[0817] DIQLTQSPSSLSASVGDRVTITCRAGESVDIFGVGFLHWYQQKPGKAPKLLIYRASNLESGVPSRFSGSGS RTDFTLTISSLQPEDFATYYCQQTNEDPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF YPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKS FNRGEC

[0818] >Bevacizumab light chain protein sequence - LC003 - SEQ ID NO: 174

[0819] DIQMTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTD FTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

[0820] >Cetuximab light chain protein sequence (LC002) - SEQ ID NO: 175

[0821] DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFT LSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

[0822] >Ipilumimab light chain protein sequence (LC008) - SEQ ID NO: 176

[0823] EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTD FTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNR GEC

[0824] The skilled person will understand that light chain antibody sequences may be combined with heavy chain sequences of their respective parental antibody type to produce an entire antibody molecule, including when the heavy chain comprises the engineering described elsewhere herein, i.e. the trastuzumab light chain may be combined with any trastuzumab heavy chain disclosed herein, including engineered trastuzumab heavy chain molecules.

[0825] Trastuzumab

[0826] In an embodiment, the agent comprises at least one light chain comprising or consisting of SEQ ID NO: 158 and at least one heavy chain comprising or consisting of SEQ ID NO: 1, 105, 2, 107, 3, 109, 4, 111, 5, 113, 6, 115, 7, 117 or 8. In an embodiment, the agent comprises two light chains and two heavy chains, wherein the light chains comprise or consist of SEQ ID NO: 158 and the heavy chains comprise or consist of SEQ ID NO: 1, 105, 2, 107, 3, 109, 4, 111, 5, 113, 6, 115, 7, 117 or 8.

[0827] Pembrolizumab

[0828] In an embodiment, the agent comprises at least one light chain comprising or consisting of SEQ ID NO: 172 and at least one heavy chain comprising or consisting of SEQ ID NO: 13, 14 or 97. In an embodiment, the agent comprises two light chains and two heavy chains, wherein the light chains comprise or consist of SEQ ID NO: 172 and the heavy chains comprise or consist of SEQ ID NO: 13, 14 or 97.

[0829] M5A

[0830] In an embodiment, the agent comprises at least one light chain comprising or consisting of SEQ ID NO: 173 and at least one heavy chain comprising or consisting of SEQ ID NO: 15. In an embodiment, the agent comprises two light chains and two heavy chains, wherein the light chains comprise or consist of SEQ ID NO: 173 and the heavy chains comprise or consist of SEQ ID NO: 15.

[0831] Bevacizumab

[0832] In an embodiment, the agent comprises at least one light chain comprising or consisting of SEQ ID NO: 174 and at least one heavy chain comprising or consisting of SEQ ID NO: 12. In an embodiment, the agent comprises two light chains and two heavy chains, wherein the light chains comprise or consist of SEQ ID NO: 174 and the heavy chains comprise or consist of SEQ ID NO: 12. Cetuximab

[0833] In an embodiment, the agent comprises at least one light chain comprising or consisting of SEQ ID NO: 175 and at least one heavy chain comprising or consisting of SEQ ID NO: 11. In an embodiment, the agent comprises two light chains and two heavy chains, wherein the light chains comprise or consist of SEQ ID NO: 175 and the heavy chains comprise or consist of SEQ ID NO: 11.

[0834] Ipilumimab

[0835] In an embodiment, the agent comprises at least one light chain comprising or consisting of SEQ ID NO: 176 and at least one heavy chain comprising or consisting of SEQ ID NO: 16. In an embodiment, the agent comprises two light chains and two heavy chains, wherein the light chains comprise or consist of SEQ ID NO: 176 and the heavy chains comprise or consist of SEQ ID NO: 16.

[0836] In some embodiments, the agent comprises light chain and / or heavy chain sequences with at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the above sequences.

[0837] In preferred embodiments, the combinations of SEQ ID NOs of the heavy chain and light chain are as per the agents that are described in the examples.

[0838] The inventors have appreciated that the technology is also generally applicable to antibody- drug-conjugates (ADCs).

[0839] Antibody-drug-conjugates (ADCs) represent a rapidly advancing category of oncology therapeutics, spanning the targeted therapy for both hematologic malignancies and solid cancers. ADCs comprise an antibody-based molecule linked to a payload, typically via a linker. The antibody-based molecule is capable of recognising specific antigens (e.g. tumour-specific antigens) and thereby allows the payload to be selectively targeted to cells of interest (e.g. cancer cells, solid tumour cells).

[0840] In a second aspect, the invention relates to an antibody-drug-conjugate (ADC) comprising the agent as described elsewhere herein, for example as described in the first aspect and one or more payloads. By "payload", we include a cytotoxic agent that can be attached to the antibody and is capable of selectively killing unwanted cells of interest (e.g. cancer cells) once the therapeutic domain binds to a tumour-specific antigen. Essentially, the payload delivers a therapeutic effect directly to the cancer cells while minimizing damage to healthy tissues. In some embodiments, a therapeutic domain comprises the payload. Suitably, the payload is linked to the therapeutic domain. "Payload" may further comprise detectable agents, for example a dye or a radioactive tracer. Alternatively, the "payload" may be replaced with a detectable agent, for example a dye or a radioactive tracer.

[0841] The one or more payload includes, but is not limited to, an a chemotherapeutic agent, an antimitotic agent (e.g. an auristatin, a maytansinoid, a calicheamicin, a tubulysin); a DNA damaging agent (e.g. a duocarmycin, a PBD dimer, a camptothecin, an anthracycline or a topoisomerase 1 inhibitor such as SN-38 or exatecan); a chemical cytotoxic agent (e.g. doxorubicin, paclitaxel, methotrexate, gemcitabine); and / or other cytotoxic agent. The one or more payloads is preferably a chemotherapeutic agent.

[0842] By "chemotherapeutic agent", we include a drug (medicament or pharmaceutically active ingredient) that has efficacy in the treatment of cancer.

[0843] The one or more payloads may be conjugated to the therapeutic domain, the cleavable domain and / or the stabilisation domain. In some embodiments, the agent may comprise a first payload attached to the therapeutic domain, cleavable domain or the stabilisation domain, and a second payload attached to a different domain selected from the therapeutic domain, cleavable domain and the stabilisation domain.

[0844] The one or more payloads may be attached to the agent via any suitable method in the art, including via native cysteine conjugation, engineered cysteine conjugation, native disulphide conjugation, native lysine conjugation, transglutaminase conjugation, glycan conjugation, engineered azide conjugation, engineered ketone conjugation, enzyme conjugation (e.g. prenyl transferase, sortase), formylglycine conjugation. Preferably the one or more payloads is attached to the agent via native or engineered cysteine conjugation.

[0845] The one or more payloads are preferably conjugated to the agent via a cysteine residue. The cysteine residue may be engineered into the agent. The cysteine residue may be native.

[0846] By "native", we include that the cysteine is not engineered (e.g. by insertion or substitution) into the therapeutic domain, cleavable domain and / or stabilisation domain as part of the conjugation process. We include that the cysteine occurs in the parental sequence of therapeutic domain, cleavable domain and / or stabilisation domain - for example, the cysteine may be present in an original (parental) antibody. In an embodiment, the one or more payloads is attached to the agent via an ADC linker. The ADC linker is preferably stable in vivo, for example in the bloodstream. The ADC linker preferably will be readily cleaved inside the target (e.g. a cancer cell or tumour) or at the target site (e.g. in or around the tumour microenvironment). Most preferably, the ADC linker will readily cleave inside the target (e.g. a cancer cell and / or tumour). The cleavage of the ADC linker at the target site may be driven by pH, enzymes, reactive thiols and / or antibody catabolism.

[0847] Examples of suitable ADC linkers are known in the art and include cleavable linkers, and include (but is not limited to):

[0848] • valine-citrulline (cleavable and protease sensitive);

[0849] • hydrozone (cleavable and acid-sensitive);

[0850] • disulphide (cleavable and glutathione-sensitive);

[0851] • N-maleimidomethylcyclohexane-l-carboxylate (MCC) (non-cleavable);

[0852] • maleimidocaproyl (non-cleavable); and / or

[0853] • mercaptoacetamidocaproyl (non-cleavable).

[0854] In a third aspect, the invention relates to a method for preparing an antibody-drug-conjugate comprising:

[0855] (a) taking an agent as defined elsewhere herein, for example as described in the first aspect; and

[0856] (b) conjugating one or more payloads to the agent, optionally via an ADC linker.

[0857] The one or more payload may be conjugated to the therapeutic domain, cleavable domain and / or stabilisation domain of the agent.

[0858] In an embodiment, step (b) comprises native cysteine conjugation, engineered cysteine conjugation, native disulphide conjugation, native lysine conjugation, transglutaminase conjugation, glycan conjugation, engineered azide conjugation, engineered ketone conjugation, enzyme conjugation (e.g. prenyl transferase, sortase) and / or formylglycine conjugation, preferably native or engineered cysteine conjugation. Additionally or alternatively, one or more of the payloads may be attached to the agent via an unnatural amino acid insertion, allowing for precise control of conjugation site, e.g. as described in Axup et al. Proc Natl Acad Sci U S A (2012) 17;109(40): 16101-16106.

[0859] The types of conjugation, ADC linkers and payloads in the methods may be as described elsewhere herein, for example as for the previous aspects of the invention.

[0860] In a fourth aspect, the invention relates to an ADC produced by the method of the third aspect. Pharmaceutical compositions

[0861] In a fifth aspect, the invention provides a pharmaceutical composition, comprising an agent according to the invention, and optionally a pharmaceutically acceptable carrier, diluent or excipient. In a sixth aspect, the invention provides a pharmaceutical composition comprising an ADC according to the invention, and optionally a pharmaceutically acceptable carrier, diluent or excipient.

[0862] The pharmaceutical composition in accordance with the invention may be administered with suitable pharmaceutically acceptable carriers, excipients, and other agents that are incorporated into formulations to provide improved transfer, delivery, tolerance, and the like. By "pharmaceutically acceptable" we include that the formulation is sterile and pyrogen free. Suitable pharmaceutically acceptable carriers, excipients or diluents are well known in the art of pharmacy. The pharmaceutically acceptable carriers, excipients or diluents must be "acceptable" in the sense of being compatible with the agent of the invention and not deleterious to the recipients thereof. Typically, the pharmaceutically acceptable carriers, excipients or diluents will be water or saline which will be sterile and pyrogen free; however, other pharmaceutically acceptable carriers, excipients or diluents may be used. Ine one embodiment, a pharmaceutically acceptable composition is manufactured according to GMP standards.

[0863] In an embodiment, the pharmaceutically acceptable carrier, diluent or excipient is a buffer, a sugar, an isotonic agent, an antioxidant, an amino acid, a chelator, a surfactant, an emulsifier, and / or saline.

[0864] By "buffer", we include a solution that can resist pH change upon the addition of an acidic or basic components. For example, the buffer may be (but is not limited to) a citrate buffer, a phosphate buffer and / or an acetate buffer.

[0865] By "isotonic agent", we include a solution that has the same concentration of solutes as another solution (e.g. as the blood). For example, the isotonic agent may be (but is not limited to) sodium chloride (e.g. saline solution or Ringer solution).

[0866] By "antioxidant", we include compounds that protect the stability and integrity of the pharmaceutical composition products by inhibiting reactions with oxygen. These compounds can act as radical scavengers, terminating oxidation reactions and reducing oxidative stress. They may play a crucial role in maintaining product quality and safety. For example, the antioxidant may be (but is not limited to) ascorbic acid or cysteine. By "chelator", we include a compound that can bind metal ions, such as ethylenediaminetetraacetic Acid (EDTA).

[0867] In an embodiment, the pharmaceutically acceptable carrier, diluent or excipient is citric acid, dextrose, acetic acid, glutamic acid, glycine, L-histidine, L-histidine monohydrochloride monohydrate, L-lysine, L-lysine hydrochloride, L-methionine, L-phenylalanine, L-threonine, mannitol, monosodium glutamate, pentetic acid, polysorbate 20, polysorbate 80, sodium acetate trihydrate, sodium chloride, sodium citrate, sorbitol, sucrose, trehalose, maltose, ethylenediaminetetraacetic Acid (EDTA), Tween, glycerol, a glycol, ethanol, fructose, glycerine, glucose and / or sucralose.

[0868] Preferably, the pharmaceutically acceptable carrier, diluent or excipient enhances solubility and / or stability of the agent. The pharmaceutically acceptable carrier, diluent or excipient may act as a cryoprotectant or lyoprotectant. Methods of determining stability or solubility of the agent in a pharmaceutical composition are routine and well known in the art. Examples include thermal shift assays, circular dichroism, differential scanning calorimetry.

[0869] Medical containers

[0870] In a seventh aspect, the invention provides a medical container comprising an agent as described elsewhere herein, for example according to the first aspect or a pharmaceutical composition as described elsewhere herein, for example according to the fifth aspect, or an antibody-drug conjugate as described elsewhere herein, for example according to the second aspect.

[0871] In an embodiment, the medical container is a vial, a syringe (e.g. a prefilled syringe), a bag or an injection device (e.g. an auto injector, a jet injector, a pump device). In an embodiment, the bag is an intravenous (IV) bag.

[0872] The bag, syringe or injection device may be for intravenous, subcutaneous, intramuscular, intradermal and / or intraosseous delivery. Preferably, the bag, syringe or injection device is for intravenous or subcutaneous delivery.

[0873] Kits

[0874] In an eighth aspect, the invention provides a kit comprising:

[0875] (i) an agent as described elsewhere herein, for example according to the first aspect or a pharmaceutical composition as described elsewhere herein, for example according to the fifth aspect, or an antibody-drug conjugate (ADC) as described elsewhere herein, for example according to the second aspect, or a medical container as described elsewhere herein, for example according to the seventh aspect; and

[0876] (ii) a label or instructions for use.

[0877] In an embodiment, the label or instructions comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number, or an authorisation number for a country of interest).

[0878] In an embodiment, the instructions indicate that the agent or pharmaceutical composition or antibody-drug conjugate or medical container is for use to treat and / or prevent a tumour in a human.

[0879] In an embodiment, the instructions indicate that the agent or pharmaceutical composition or antibody-drug conjugate or medical container is for use to treat and / or prevent a cancer in a human. Preferably the cancer is a solid tumour. The cancer may be as defined elsewhere in the application.

[0880] In an embodiment, the kit further comprises an IV bag, syringe or injection device that comprises the agent.

[0881] By "label", we include a written, printed or graphic matter that accompanies the agent, pharmaceutical composition, antibody-drug conjugate or medical container which seeks to identify the agent by any means, e.g. by name, INN nomenclature, a trade name etc.

[0882] By "instructions", we include a display of written, printed or graphic matter on a container or on or in packaging of an article, for example the written material displayed on a medical container containing the agent, or composition, or antibody-drug conjugate, or details regarding the composition and use of the agent or antibody-drug conjugate included in a kit containing the agent or the composition or the antibody-drug conjugate. Instructions set forth the method of the treatment as contemplated to be administered or performed.

[0883] In an embodiment, the kit further comprises packaging. By "packaging" we include components for organising and / or containing the agents or compositions or medical containers or antibodydrug conjugates for storage, distribution and use. Packaging can include, but is not limited to, boxes, bags etc.

[0884] Therapeutic uses

[0885] The agent, ADC, pharmaceutical composition, medical container or kit is suitable for use as a medicament, such as in therapy, such as in the treatment or prevention of a tumour, optionally of a cancer. By 'treatment' we include both therapeutic and prophylactic treatment of the patient. The term 'prophylactic' is used to encompass the use of an agent, ADC, formulation, pharmaceutical composition, medical container or kit, as described herein which either prevents or reduces the likelihood of cancer, or the spread, dissemination, or metastasis of localised cancer in a patient or subject. The term 'prophylactic' also encompasses the use of an agent, ADC, formulation, pharmaceutical composition, medical container or kit, as described herein to prevent recurrence of cancer in a patient who has previously been treated for cancer.

[0886] As used herein, the terms "treatment" or "treating" denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result. Such beneficial or desired clinical results include, but are not limited to, one or more of the following : reducing the proliferation of (or destroying) cancerous cells or other diseased cells, reducing metastasis of cancerous cells found in cancers, shrinking the size of the tumour, decreasing symptoms resulting from the disease, increasing the quality of life of those suffering from the disease, decreasing the dose of other medications required to treat the disease, delaying the progression of the disease, and / or prolonging survival of individuals.

[0887] Upon binding, the agent (or ADC) may directly exert a therapeutic effect (e.g. inducing cell death via Antibody Dependent Cellular Cytotoxicity (ADCC), complement dependent cytotoxicity (CDC) or by virtue of carrying a radioisotope or other cytotoxic moiety). Alternatively, the bound agent may serve as a diagnostic (imaging) tool and may guide the choice of therapy or aid surgical removal of the unwanted cells.

[0888] In a particularly preferred embodiment, the cleavage site is selectively cleaved outside of the unwanted cell, at or near its surface, so that the therapeutic domain is released without the entire agent needing to be internalised.

[0889] Suitably the vicinity of unwanted cells or the tumour comprises a high titre and / or a high activity of tumour-specific proteases.

[0890] The titre and / or activity of a protease in the vicinity of unwanted cells or the tumour will be considered "high" if the titre and / or activity of the protease is significantly higher than titre and / or activity of the protease at other locations considered further than the vicinity of unwanted cells of the tumour. Those skilled in the art would be capable of selecting an appropriate assay to measure titre and activity of the protease, for example ELISA, activitybased protein profiling (ABPP), mass spectrometry, fluorescent probe imaging. In one embodiment, protease activity is measured by an ELISA assay. Suitably, the subject of treatment or prevention is a mammal, preferably a human. In some embodiments, the subject is a non-human mammal, such as a rodent, a non-human primate, companion animal (e.g. cat, dog, horse), farm animal, work animal, zoo animal.

[0891] Suitably, the tumour is a solid tumour. In some embodiments, the cancer is selected from the group consisting of: Adenocarcinoma, Adenosarcoma, Adrenal cancer, Adrenocortical carcinoma, Anal cancer, Anaplastic astrocytoma, Angiosarcoma, Appendix cancer, Astrocytoma, Basal cell carcinoma, B-Cell lymphoma, Bile duct cancer, Bladder cancer, Bone cancer, Bowel cancer, Brain cancer, Brain stem glioma, Brain tumour, Breast cancer, Carcinoid tumours, Cervical cancer, Cholangiocarcinoma, Chondrosarcoma, Colon cancer, Colorectal cancer, Craniopharyngioma, Cutaneous melanoma, Diffuse astrocytoma, Ductal carcinoma in situ, Endometrial cancer, Ependymoma, Epithelioid sarcoma, Esophageal cancer, Ewing sarcoma, Extrahepatic bile duct cancer, Eye cancer, Fallopian tube cancer, Fibrosarcoma, Gallbladder cancer, Gastric cancer, Gastrointestinal cancer, Gastrointestinal carcinoid cancer, Gastrointestinal stromal tumours, General, Germ cell tumour, Glioblastoma multiforme, Glioma, Head and neck cancer, Hemangioendothelioma, Hodgkin's disease Hypopharyngeal cancer, Infiltrating ductal carcinoma, Infiltrating lobular carcinoma, Inflammatory breast cancer, Intestinal Cancer, Intrahepatic bile duct cancer, Invasive I infiltrating breast cancer, Islet cell cancer, Jaw cancer, Kaposi sarcoma, Kidney cancer, Laryngeal cancer, Leiomyosarcoma, Leptomeningeal metastases Lip cancer, Liposarcoma, Liver cancer, Lobular carcinoma in situ, Low-grade astrocytoma, Lung cancer, Lymph node cancer, Lymphoma, Male breast cancer, Medullary carcinoma, Medulloblastoma, Melanoma, Meningioma, Merkel cell carcinoma, Mesenchymal chondrosarcoma, Mesenchymous, Mesothelioma, Metastatic breast cancer, Metastatic melanoma, Metastatic squamous neck cancer, Mixed gliomas, Mouth cancer, Mucinous carcinoma, Mucosal melanoma, Multiple myeloma, Nasal cavity cancer, Nasopharyngeal cancer, Neck cancer, Neuroblastoma, Neuroendocrine tumours, Non-small cell lung cancer, Oat cell cancer, Ocular cancer, Ocular melanoma, Oligodendroglioma, Oral cancer, Oral cavity cancer, Oropharyngeal cancer, Osteogenic sarcoma, Osteosarcoma, Ovarian cancer, Ovarian epithelial cancer, Ovarian germ cell tumour, Ovarian primary peritoneal carcinoma, Ovarian sex cord stromal tumour, Paget's disease, Pancreatic cancer, Papillary carcinoma, Paranasal sinus cancer, Parathyroid cancer, Pelvic cancer, Penile cancer, Peripheral nerve cancer, Peritoneal cancer, Pharyngeal cancer, Pheochromocytoma, Pilocytic astrocytoma, Pineal region tumour, Pineoblastoma, Pituitary gland cancer, Primary central nervous system lymphoma, Prostate cancer, Rectal cancer, Renal cell cancer, Renal pelvis cancer, Rhabdomyosarcoma, Salivary gland cancer, Sarcoma, Sarcoma, bone, Sarcoma, soft tissue, Sarcoma, uterine, Sinus cancer, Skin cancer, Small cell lung cancer, Small intestine cancer, Soft tissue sarcoma, Spinal cancer, Spinal column cancer, Spinal cord cancer, Spinal tumour, Squamous cell carcinoma, Stomach cancer, Synovial sarcoma, T-cell lymphoma, Testicular cancer, Throat cancer, Thymoma / thymic carcinoma, Thyroid cancer, Tongue cancer, Tonsil cancer, Transitional cell cancer, Transitional cell cancer, Transitional cell cancer, Triple-negative breast cancer, Tubal cancer, Tubular carcinoma, Ureteral cancer, Ureteral cancer, Urethral cancer, Uterine adenocarcinoma, Uterine cancer, Uterine sarcoma, Vaginal cancer, and Vulvar cancer.

[0892] In a further aspect, there is a method for the treatment or prevention of a tumour, optionally of cancer, wherein the method comprises one or more (e.g. all) of the following steps: a) obtaining (or having obtained) a sample of the tumour and / or the vicinity of the tumour; b) determining (or having determined) one or more proteases expressed by the tumour and / or present in the vicinity of the tumour; c) administering to the subject an agent (or ADC) wherein the cleavable domain is cleavable by the one or more proteases determined to be expressed by said tumour and / or present in said vicinity of the tumour (or a pharmaceutical composition comprising said agent or ADC).

[0893] The term "sample" includes any biological sample from the individual, to be tested in the methods and uses of the invention. It will be appreciated that the sample may comprise one or more tissue, cell and / or biological fluid taken from (such as isolated from) the individual (e.g., blood; serum; plasma; serum plasma; urine; saliva; intestinal cells; biopsy; stool).

[0894] By "determining one or more proteases expressed by the tumour and / or present in the vicinity of the tumour", we include the meaning of determining whether or not the sample contains one or more proteases expressed by the tumour and / or present in the vicinity of the tumour. Preferably, this comprises exposing the agent (or ADC) to the sample and determining which proteases are expressed and / or present.

[0895] In yet a further aspect, the invention provides a method of treating or preventing cancer, wherein the method comprises administering one or more agents, ADCs or pharmaceutical compositions according to the invention.

[0896] Preferably, the one or more agents, ADCs or pharmaceutical compositions is administered by injection or infusion. However, in practice it can be administered by any suitable means. Those skilled in the art would be capable of selecting an appropriate route of administration. Preferably the one or more agents, ADCs or pharmaceutical compositions are administered intravenously or subcutaneously. Most preferably, the one or more agents, ADCs or pharmaceutical compositions are administered intravenously.

[0897] In yet another aspect, the invention provides the agent, ADC or pharmaceutical composition according to the invention for use in preventing or treating a condition characterised by the presence of unwanted cells, optionally wherein the condition is cancer (i.e. the unwanted cells are tumour cells). By a "condition characterised by the presence of unwanted cells" we include any biological or medical condition or disorder in which at least part of the pathology is mediated by the presence of unwanted cells. The condition may be caused by the presence of the unwanted cells or else the presence of the unwanted cells may be an effect of the condition. Examples of particular conditions include tumours (benign or malignant), autoimmune conditions, cardiovascular diseases, degenerative diseases, diabetes, allergic disease (e.g. asthma), neurodegenerative diseases such as Alzheimer's, transplantation patients and infectious diseases. It will be appreciated that the agent also has utility in regenerative medicine (e.g. laboratory grown organs or tissues). It is particularly preferred if the condition is a tumour (e.g. a malignant disease) and the unwanted cells are tumour cells or tumour-associated tissue.

[0898] In another aspect, the invention provides a method of improving the penetrability of a therapeutic domain into a tissue or tumour, wherein the method comprises engineering an agent comprising: i) a therapeutic domain; ii) a cleavable domain; and iii) a stabilisation domain. The therapeutic domain, cleavable domain and stabilisation domain may be as described elsewhere herein.

[0899] In another aspect, the invention provides a method of improving the penetrability of a therapeutic domain into a tissue or tumour, wherein the method comprises engineering an ADC comprising: i) a therapeutic domain; ii) a cleavable domain; iii) a stabilisation domain, and iv) one or more payloads. The therapeutic domain, cleavable domain, stabilisation domain and payload may be as described elsewhere herein.

[0900] By "engineering an agent", we include the meaning of genetic modification. Such genetic modification could include (i) mutations of nucleotide bases of the agent (such as one or more addition, one or more deletion, one or more substitution, or a combination thereof); and (ii) expression (such as recombinant expression) in a cell culture system (such as mammalian cells, bacteria, yeast or insect cells).

[0901] In yet another aspect, the invention provides use of the agent, an ADC or a pharmaceutical composition according to the invention in the manufacture of a medicament for the treatment or prevention of a tumour, optionally for the treatment or prevention of cancer. A subject may be treated with a single dose, or multiple doses, of an effective amount of the agents, ADCs or pharmaceutical compositions of the invention. Where multiple administrations are made, these may be made at a rate of, for example, once, twice, three times, four times or more often per day, week or month, and may be continued for a period of time necessary and effective obtain a therapeutically or prophylactically beneficial effect. For example, treatment may continue for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more days, weeks, months or years, or even for the rest of the life of the subject. The amount of the agent which is administered to the individual is an amount effective to combat the particular individual's condition. In some embodiments, the agent, ADC or pharmaceutical composition is administered in combination with one or more additional agents, such as a chemotherapeutic agent, an immunotherapeutic agent or a radiotherapeutic agent. In some embodiments, the agents, ADCs or pharmaceutical compositions of the invention and the additional agent(s) are formulated in a single composition. In an alternative embodiment, the agent, ADC or pharmaceutical composition of the invention and the additional agent(s) are administered as two or more separate compositions. In yet another embodiment, the agent, ADC or pharmaceutical composition of the invention and the additional agent(s) are administered simultaneously. In an alternative embodiment, the agent, ADC or pharmaceutical composition of the invention and the additional agent(s) are administered sequentially.

[0902] It may be appropriate to administer a particular protease inhibitor so as to improve the target selectivity of the agent or ADC of the invention. For example, if a therapeutic domain is known to bind cells in both the heart and breast tissue, but only those in the breast are to be targeted, it may be desirable to administer an additional agent / inhibitor that selectively inhibits the protease in the heart but not the breast.

[0903] In other words, an additional agent / inhibitor is administered to inhibit a protease that resides in the vicinity of wanted cells but not in the vicinity of unwanted cells. This is particularly useful in the event that a cleavable domain of the agent or ADC of the invention is cleavable by multiple enzymes, some of which reside in the vicinity of unwanted cells and some of which reside in the vicinity of wanted cells. In this case, targeting specificity may be improved by administering a protease inhibitor that inhibits a protease that resides in the vicinity of wanted cells but nevertheless is capable of cleaving the cleavable domain and therefore releasing the therapeutic domain of the agent or ADC of the invention. The effect of administering the inhibitor would be to ensure that the therapeutic domain is preferentially released in the vicinity of the unwanted cells.

[0904] In yet another aspect, the invention provides a method of improving the efficacy of an agent, the method comprising engineering the agent to comprise: i) a therapeutic domain; ii) a cleavable domain; and iii) a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. Any feature described herein may be used in this method and / or agent.

[0905] In yet another aspect, the invention provides a method of improving the efficacy of an ADC, the method comprising engineering the ADC to comprise: i) a therapeutic domain; ii) a cleavable domain; iii) a stabilisation domain, and iv) one or more payloads, wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. Any feature described herein may be used in this method and / or agent. Small agents may offer advantageous pharmacokinetic properties (such as a better ability to penetrate solid tumours, higher tumour uptake levels, improved specificity and / or efficacy, more predictable, more linear elimination). However, small agents also carry significant disadvantages such as an ability to be rapidly eliminated (via the GFB for example), short halflives etc.

[0906] To mitigate the disadvantages of small agents, the inventor has created engineered agents to contain i) a therapeutic domain; ii) a cleavable domain; and iii) a stabilisation domain, and optionally (iv) one or more payloads and optionally one or more ADC linker(s). This makes use of the advantages of larger agents (slow elimination and long half-lives) until the agent comes into contact with tumour-specific proteases in the vicinity of solid tumours, where cleavage of the agent causes reduction in the size of the agent. In the TME, the smaller parts of the agent (stabilisation domain and therapeutic domain) benefit from the advantageous pharmacokinetic properties discussed above.

[0907] The method according to the aspect above, wherein the improved efficacy of the agent is manifest by achieving the desired clinical result of the agent (such as reducing the proliferation of, destroying, reducing metastasis of cancerous cells or other diseased cells, shrinking the size of the tumour, decreasing symptoms resulting from the disease, increasing the quality of life of the subject, delaying the progression of the disease, and / or prolonging survival of the subject) using the same dosing regimen of the agent. Those skilled in the art would be capable of selecting an appropriate assay to measure the desired clinical result of the agent. For example, surgical removal of the solid tumour from the subject to measure the size of the tumour or fluorescent imaging of an in vivo tumour in a cancer animal model to visualise cell proliferation.

[0908] In another aspect, the invention provides a method of improving the specificity of an agent, the method comprising engineering the agent to comprise: i) a therapeutic domain; ii) a cleavable domain; and iii) a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example according to the first aspect and / or may be comprised by a pharmaceutical composition as described elsewhere herein, for example according to the fifth aspect.

[0909] In another aspect, the invention provides a method of improving the specificity of an ADC, the method comprising engineering the ADC to comprise: i) a therapeutic domain; ii) a cleavable domain; iii) a stabilisation domain; and iv) one or more payloads, wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The ADC may be as defined elsewhere herein, for example according to the second aspect. In the method according to either of the two aspects above the improved specificity of the agent or ADC may be manifest by a higher titre of the therapeutic domain (and optionally the one or more payload) in diseased tissue compared to the titre of the therapeutic domain in non-diseased tissue. Those skilled in the art would be capable of selecting an appropriate assay to measure titre of the therapeutic domain in tissue. For example, biopsies from diseased tissue and non-diseased tissue could be taken. The titre of the therapeutic domain in each sample could then be assessed by immunostaining using antibodies (such as anti-CHl antibodies, or anti-CLl antibodies) or by ELISA.

[0910] The method may further comprise: i. applying (or administering) the agent (or ADC) of the invention or a pharmaceutical composition comprising an agent (or ADC); ii. the cleavable domain being cleaved at the tumour; and iii. the therapeutic domain separating from the stabilisation domain. The terms "at the tumour" and "in the vicinity of the tumour" are used interchangeably. The agent may be as defined elsewhere herein, for example in according to the first aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the fifth or sixth aspect. The ADC may be as described elsewhere herein, for example as defined according to the second aspect.

[0911] In yet another aspect, the invention provides use of an agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain or a pharmaceutical composition comprising an agent to improve the penetrability of the therapeutic domain into a tumour. The agent may be as defined elsewhere herein, for example in according to the first aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the fifth aspect.

[0912] In yet another aspect, the invention provides use of an ADC comprising: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain and iv. one or more payloads or a pharmaceutical composition comprising an ADC to improve the penetrability of the therapeutic domain into a tumour. The ADC may be as defined elsewhere herein, for example according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect.

[0913] In another aspect, the invention provides use of an agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain, or a pharmaceutical composition comprising an agent as described elsewhere herein, to reduce the size of the agent at a tumour site. It will be appreciated that the size of the agent is reduced by specific cleavage of the cleavage site within the cleavable domain. Cleavage of the agent at the tumour causes the formation of multiple smaller portions of the agent, which may comprise the stabilisation domain and therapeutic domain. The agent may be as described elsewhere herein, for example as defined in according to the first aspect and / or may be comprised by a pharmaceutical composition as described elsewhere herein, for example according to the fifth aspect.

[0914] In another aspect, the invention provides use of an ADC comprising: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and iv. one or more payloads and optionally one or more ADC linkers, to reduce the size of the ADC at a tumour site. It will be appreciated that the size of the ADC is reduced by specific cleavage of the cleavable domain. Cleavage of the ADC at the tumour causes the formation of multiple smaller portions of the ADC, which may comprise the stabilisation domain and therapeutic domain and optionally one or more payloads (for example if the ADC linker is also cleaved by a protease at the tumour). The ADC may be as defined elsewhere herein, for example according to the second aspect.

[0915] In yet another aspect, the invention provides use of an agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain or a pharmaceutical composition comprising an agent to improve the efficacy of the agent wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example in according to the first aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the fifth aspect.

[0916] In yet another aspect, the invention provides use of an ADC comprising: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and iv. one or more payloads and optionally one or more ADC linkers to improve the efficacy of the ADC wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The ADC may be as described elsewhere herein, for example as defined according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect.

[0917] In another aspect, the invention provides use of an agent comprising: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain or a pharmaceutical composition comprising an agent to improve the specificity of the agent wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example according to the first aspect and / or may be comprised by a pharmaceutical composition described elsewhere herein, for example according to the fifth aspect.

[0918] In another aspect, the invention provides use of an ADC comprising: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and iv. one or more payloads and optionally one or more ADC linkers to improve the specificity of the ADC wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The ADC may be as defined elsewhere herein, for example according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect.

[0919] In yet another aspect, the invention provides use of a cleavable domain to improve the penetrability of a therapeutic domain into a tumour, the use comprising engineering an agent to comprise: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example according to the first aspect and / or may be comprised by a pharmaceutical composition described elsewhere herein, for example according to the fifth aspect.

[0920] In yet another aspect, the invention provides use of a cleavable domain to improve the penetrability of a therapeutic domain and / or one or more payloads into a tumour, the use comprising engineering an ADC to comprise: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and / or iv. one or more payloads and optionally one or more ADC linkers, wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The one or more payloads may be attached (e.g. directly) to the therapeutic domain, and / or attached to the therapeutic domain following cleavage of the cleavable domain. The ADC may be as defined elsewhere herein, for example according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect.

[0921] In another aspect, the invention provides use of a cleavable domain to reduce the size of the agent at a tumour site, the use comprising engineering an agent to comprise: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example according to the first aspect and / or may be comprised by a pharmaceutical composition described elsewhere herein, for example according to the fifth aspect.

[0922] In another aspect, the invention provides use of a cleavable domain to reduce the size of the ADC at a tumour site, the use comprising engineering an ADC to comprise: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and iv. one or more payloads and optionally one or more ADC linkers, wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The one or more payloads may be attached (e.g. directly) to the therapeutic domain and / or attached to the therapeutic domain following cleavage or the cleavable domain. The ADC may be as defined elsewhere herein, for example according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect. In yet another aspect, the invention provides use of a cleavable domain to improve the efficacy of an agent, the use comprising engineering the agent to comprise: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example according to the first aspect and / or may be comprised by a pharmaceutical composition described elsewhere herein, for example according to the fifth aspect.

[0923] In yet another aspect, the invention provides use of a cleavable domain to improve the efficacy of an ADC, the use comprising engineering the ADC to comprise: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and iv. one or more payloads and optionally one or more ADC linkers, wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The one or more payloads may be attached (e.g. directly) to the therapeutic domain, and / or attached to the therapeutic domain following cleavage or the cleavable domain. The ADC may be as defined elsewhere herein, for example according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect.

[0924] In another aspect, the invention provides use of a cleavable domain to improve the specificity of an agent, the use comprising engineering the agent to comprise: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The agent may be as defined elsewhere herein, for example according to the first aspect and / or may be comprised by a pharmaceutical composition described elsewhere herein, for example according to the fifth aspect.

[0925] In another aspect, the invention provides use of a cleavable domain to improve the specificity of an ADC, the use comprising engineering the ADC to comprise: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain, and iv. one or more payloads and optionally one or more ADC linkers, wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The one or more payloads may be attached (e.g. directly) to the therapeutic domain, and / or attached to the therapeutic domain following cleavage or the cleavable domain. The ADC may be as described elsewhere herein, for example as defined according to the second aspect and / or may be comprised by a pharmaceutical composition as defined elsewhere herein, for example according to the sixth aspect.

[0926] In an embodiment, the improved specificity of the agent is manifest by a higher titre of the therapeutic domain in diseased tissue compared to the titre of the therapeutic domain in nondiseased tissue. Preferably, the agent is as described elsewhere herein, for example the agent is according to the first aspect of the invention wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain.

[0927] In an embodiment, the improved specificity of the ADC is manifest by a higher titre of the therapeutic domain and / or the one or more payloads in diseased tissue compared to the titre of the therapeutic domain in non-diseased tissue. Preferably, the ADC is as described elsewhere herein, for example the ADC is according to the second aspect of the invention wherein the cleavable domain is positioned between the therapeutic domain and the stabilising domain. The one or more payloads may be (e.g. directly) attached to the therapeutic domain, and / or attached to the therapeutic domain following cleavage or the cleavable domain.

[0928] In yet another aspect, the invention provides an agent (e.g. the agent according to the first aspect of the invention), a pharmaceutical composition, a medical container, an ADC or a kit (each as described elsewhere herein) for use in diagnosing a disease or disorder. By "diagnosis" we include the detection of cancer cells, either in vivo (i.e. within the body of a patient) or ex vivo (i.e. within a tissue or cell sample removed from the body of a patient). Those skilled in the art would be capable of selecting an appropriate assay to detect the cancer cells. As will be appreciated and as discussed herein, the agent, the therapeutic domain, the stabilisation domain may be detected by labelling (such as labelling the Fab region of an antibody with a fluorophore) and subsequent detection of the label (such as detection of the fluorophore using an antibody), by immunostaining using antibodies (such as anti-CHl antibodies, anti-lambda antibodies, or anti-kappa antibodies) or by ELISA.

[0929] In another aspect of the invention the use comprises: i. administering the agent or the pharmaceutical composition comprising the agent to a subject; ii. the cleavable domain being cleaved at the tumour (or in the TME); and iii. the therapeutic domain separating from the stabilisation domain; iv. increased accessibility to the cells for binding of the therapeutic domain compared to if cleavage and separation according to steps (ii) and (iii) had not occurred. By "accessibility to the cells for binding", we include the meaning that some cells that previously had a limited ability to be accessed and be bound by parts of the agent (such as therapeutic domain or stabilisation domain) would be more accessible and more able to be bound by the parts of the agent. For example, prior to step (ii), the parts of the agent would bind to a limited extent to the interior parts of a tumour (e.g. in the TME). After step (iii), the parts of the agent would bind to more of the interior parts of a tumour (e.g. in the TME). Those skilled in the art would be capable of selecting an appropriate assay to assess accessibility to the cells for binding of the therapeutic domain. For example, the same methods used for measuring the ability of the agent to penetrate a solid tumour discussed above may be used. Particularly preferably, selective cleavage of the cleavable domain enables the release of the therapeutic domain at or near to the cell surface of the unwanted cells. The terms "near to" and "in the vicinity of the tumour" are used interchangeably.

[0930] In another aspect of the invention the use comprises: i. administering the ADC or a pharmaceutical composition comprising the ADC to a subject; ii. the cleavable domain being cleaved at the tumour or in the tumour microenvironment; and iii. the therapeutic domain separating from the stabilisation domain (preferably with one or more payloads attached thereto); iv. increased accessibility to the cells for binding of the therapeutic domain compared to if cleavage and separation according to steps (ii) and (iii) had not occurred. By "accessibility to the cells for binding", we include the meaning that some cells that previously had a limited ability to be accessed and be bound by parts of the ADC (such as therapeutic domain or stabilisation domain) would be more accessible and more able to be bound by the parts of the ADC. For example, prior to step (ii), the parts of the ADC would bind to a limited extent to the interior parts of a tumour or within the tumour microenvironment. After step (iii), the parts of the ADC would bind to more of the interior parts of a tumour or within the tumour microenvironment. Those skilled in the art would be capable of selecting an appropriate assay to assess accessibility to the cells for binding of the therapeutic domain. For example, the same methods used for measuring the ability of the agent to penetrate a solid tumour discussed above may be used.

[0931] In another aspect, the invention relates to a method of determining whether a subject (in particular a cancer subject or a subject who has been diagnosed with a solid tumour) is suitable for being treated by an agent, comprising the steps of:

[0932] (a) having a serum sample or biopsy sample obtained from the subject;

[0933] (b) detecting (or having detected) whether at least one cell in the serum sample or biopsy sample comprises a predetermined antigen of interest and / or a predetermined protease of interest, and wherein the agent comprises:

[0934] (i) a therapeutic domain that specifically binds the antigen of interest;

[0935] (ii) a cleavable domain comprising a cleavage site that is capable of being specifically cleaved by the protease of interest; and

[0936] (iii) a stabilisation domain, optionally wherein the agent is an ADC which further comprises one or more payloads and optionally one or more ADC linkers.

[0937] Preferably, step (b) comprises determining (or having determined) whether the sample comprises the antigen of interest and the protease of interest.

[0938] Methods for detecting whether at least one cell in a serum or biopsy sample contains an antigen are well known in the art, and include enzyme-linked immunosorbent assays (ELISAs) and lateral flow immunoassays (LFIAs). Methods for determining whether a sample contains protease are well known in the art, and include fluorescent assays (e.g. using fluorogenic substrates); western blotting, ELISA, colorimetric assays etc.

[0939] The method may further comprise a step of obtaining (or having obtained) a serum sample or biopsy sample from a subject prior to step (a). Methods for performing biopsies are well known in the art, e.g. needle biopsy, punch biopsy, endoscopic biopsy, excisional biopsy and / or perioperative biopsy. A serum sample can be obtained, for example, by a standard blood test using a needle or cannula.

[0940] The method may further include a step (c) determining (or having determined) that the subject is suitable for treatment if (i) the target antigen of interest is present in the serum sample or biopsy sample and / or (ii) the protease of interest is present in the serum sample or biopsy sample. Preferably step (c) comprises determining (or having determined) that the subject is suitable for treatment if (i) the target antigen of interest is present in the serum sample or biopsy sample and (ii) the protease of interest is present in the serum sample or biopsy sample. The subject may be informed of the result. The method may comprise generating a report which is provided to the subject or medical personnel involved in the care of the subject.

[0941] The method may further comprise step (d) administering the subject the agent. The method may further comprise step (d) treating and / or preventing cancer in the subject by administering the subject the agent.

[0942] In yet another aspect, the invention provides a method for introducing a therapeutic domain into a tumour microenvironment in a subject, wherein the method comprises:

[0943] • engineering an agent or ADC as described elsewhere herein, wherein the agent comprises a cleavage site;

[0944] • selectively cleaving the cleavage site outside of tumour or at the tumour surface and / or near the tumour surface, thereby releasing the therapeutic domain from the stabilisation domain;

[0945] • introducing the therapeutic domain into the tumour microenvironment via diffusion.

[0946] The reduction of size in the vicinity of the tumour microenvironment may improve the penetrability of the therapeutic domain into the tumour.

[0947] In yet another aspect, the invention provides a method for reducing the size of an agent or ADC in vivo, comprising:

[0948] • engineering and / or providing an agent or ADC as described elsewhere herein, wherein the agent or ADC comprises a cleavage site;

[0949] • selectively cleaving the cleavage site in vivo. In yet another aspect, the invention relates to a method for modifying a parental antibody to form an engineered agent, the method comprising engineering a cleavable domain (in particular a cleavage site and one or more linker domain(s)) into the hinge region (in particular the upper or lower hinge region) of the parental antibody. The cleavable domain, agent, and hinge region position may be as described elsewhere herein. The method may include a step of expressing the engineered agent. The method may include a step of purifying the engineered agent. The method may include a step of formulating the engineered agent into a pharmaceutically acceptable formulation. The method may include a step of packaging the pharmaceutical formulation into a medical container and optionally providing instructions for use in or with the packaging. The engineered agent or ADC may be used in any of the methods or uses described elsewhere herein.

[0950] In yet another aspect, the invention relates to the use of a cleavable domain in engineering a parental antibody (or parental ADC) to form an agent or ADC as described elsewhere herein, wherein the cleavable domain (in particular a cleavage site and one or more linker domain(s)) is introduced into the hinge region (in particular the upper or lower hinge region) of the parental antibody or ADC.

[0951] A method of engineering an agent or ADC, comprising: a) identifying a protease site wherein the protease is a protease which is preferentially expressed or is preferentially active in a tumour or tumour microenvironment; b) engineering an agent or ADC as described elsewhere herein, wherein the cleavable domain comprises the protease site from step (a) as a cleavage site.

[0952] The protease site is as described elsewhere herein. The protease site may be identified by any suitable method in the art, e.g. by mining literature and / or by artificial intelligence or machine learning - alone or in combination with wet lab work.

[0953] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or the specification may mean "one," but it is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0954] Herein, when "about" is applied to a value or parameter, it includes (and describes) embodiments involving the value or parameter itself. For example, a description referring to "about X" includes a description of "X". A numerical range includes the numbers defining the range. Although the numerical ranges and parameters setting forth the broad scope of the present disclosure are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. However, any numerical values inherently contain certain errors necessarily resulting from the standard deviation found in their respective tests or measurements. Moreover, all ranges disclosed herein should be understood to encompass any and all subranges subsumed therein.

[0955] These, and other, embodiments of the invention will be better appreciated and understood when considered in conjunction with the description and the accompanying drawings. It should be understood, however, that the description, while indicating various embodiments of the invention and numerous specific details thereof, is given by way of illustration and not of limitation. Many substitutions, modifications, additions and / or rearrangements may be made within the scope of the invention without departing from the spirit thereof, and the invention includes all such substitutions, modifications, additions and / or rearrangements, i.e. all parts and features of this description, aspect and embodiments are intended to be combinable with each other, unless it is apparent to the skilled person that such a combination is not technically possible.

[0956] The listing or discussion in this specification of an apparently prior-published document should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

[0957] EXAMPLES

[0958] Example 1: Antibody Designs

[0959] A cancer specific protease cleavage site (LSGRSDNH (SEQ ID NO: 77), emboldened and underlined below) for uPA was engineered in different regions of an exemplary antibody, trastuzumab, to enable physical separation of the therapeutic domain and the stabilisation domain at the tumour (Figure 6).

[0960] The protease site was engineered into the upper hinge region of the trastuzumab. The C- terminal cysteine from the CHI domain remained intact, and insertions and / or substitutions were added after this residue up until the first cysteine of the middle hinge region. Structurally, this is defined in IgGl as:

[0961] Table A

[0962] Table B

[0963] The skilled person would be able to determine the equivalent positions in other antibody isotypes.

[0964] Seven different designs that included additions or substitutions of different regions of the antibody to the protease cleavage site are as follows:

[0965] In the engineered trastuzumab antibody design 1 (DI), the cleavage site LSGRSDNH (SEQ ID NO: 77) was added after the cysteine residue that ends the CHI domain and prior to the DKTHT (SEQ ID NO: 78) hinge region (bold and italicised). GS was added as an additional N terminal 2 amino acid linker domain to allow for greater accessibility of the protease. These were inserted after position 5 (IMGT hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering). These were added before residues 6 (IMGT hinge numbering), 221 (EU numbering) and 234 (Kabat and Chothia numbering).

[0966] Engineered trastuzumab antibody design 1, DI (SEQ ID NO: 105)

[0967] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSLSGRSDNHASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCV VVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAP IEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK In the engineered trastuzumab antibody design 2 (D2), the cleavage site LSGRSDNH (SEQ ID NO: 77) was added after the cysteine residue that ends the CHI domain and prior to the DKTHT (SEQ ID NO: 78) hinge region (bold and italicised). No linker domain amino acids were added. These were inserted after position 5 (IMGT hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering). These were added before residues 6 (IMGT hinge numbering), 221 (EU numbering) and 234 (Kabat and Chothia numbering).

[0968] Engineered trastuzumab antibody design 2, D2 (SEQ ID NO: 107)

[0969] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCLSGRSDNHASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFF LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0970] In the engineered trastuzumab antibody design 3 (D3), the cleavage site LSGRSDNH (SEQ ID NO: 77) was added after the cysteine residue that ends the CHI domain and prior to the DKTHT (SEQ ID NO: 78) hinge region (bold and italicised). GS was added as an additional 2 amino acid linker domains C and N terminal to the protease cleavage site for greater accessibility. These were inserted after position 5 (IMGT hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering). These were added before residues 6 (IMGT hinge numbering), 221 (EU numbering) and 234 (Kabat and Chothia numbering).

[0971] Engineered trastuzumab antibody design 3, D3 (SEQ ID NO: 109)

[0972] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSLSGRSDNHGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVT CVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALP APIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDS DGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0973] In the engineered trastuzumab antibody design 4 (D4), the DKTHT (SEQ ID NO: 78) hinge region was substituted with LSGRSDNH (SEQ ID NO: 77) after the cysteine residue that ends the CHI domain and prior to the hinge disulphide bond region (CPPC (SEQ ID NO: 79)). This is the minimal sequence modification needed to insert a protease cleavage site. These were substituted after position 5 (IMGT hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering), and positions 6-10 (IMGT hinge numbering) or 221-225 (EU numbering) or 234-238 Kabat and Chothia numbering were deleted and replaced by the UpA cleavage site). Engineered trastuzumab antibody design 4, D4 (SEQ ID NO: 111)

[0974] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCLSGRSDNHCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0975] In the engineered trastuzumab antibody design 5 (D5), the DKTHT (SEQ ID NO: 78) hinge region was substituted with LSGRSDNHT (SEQ ID NO: 80) after the cysteine residue that ends the CHI domain and prior to the hinge disulphide bond region (CPPC (SEQ ID NO: 79)). The additional threonine was thought to provide a similar neighbouring environment to the cysteine of the hinge region to allow that region to fold correctly. Design 5 adds a glycosylation site to the protease cleavage site that would not allow cleavage with the uPA protease. These were substituted after position 5 (IMGT hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering), and positions 6-10 (IMGT hinge numbering) or 221-225 (EU numbering) or 234-238 Kabat and Chothia numbering were deleted and replaced by the UpA cleavage site).

[0976] Engineered trastuzumab antibody design 5, D5 (SEQ ID NO: 113)

[0977] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCLSGRSDNHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0978] In the engineered trastuzumab antibody design 6 (D6), LSGRSDNH (SEQ ID NO: 77) was added to the CH2 domain two amino acids (PA) after the hinge disulphide bond region (CPPC (SEQ ID NO: 79)) (to give a F(ab')2 fragment) to allow accessibility of the protease. The protease cleavage site as added after residue 1.6 of the IMGT unique numbering for the C- domain, residue 231 according to the EU numbering scheme and 244 according to the Kabat and Chothia numbering schemes. The protease cleavage site was also added before residue 1.5 from the IMGT unique numbering for the C-domain, residue 232 according to the EU numbering scheme and 245 according to the Kabat and Chothia numbering schemes.

[0979] Engineered trastuzumab antibody design 6, D6 (SEQ ID NO: 115) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI

[0980] SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS

[0981] GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP

[0982] SNTKVDKRVEPKSCDKTHTCPPCPALSGRSDNHPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS

[0983] HEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTIS

[0984] KAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYS

[0985] KLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0986] In the engineered trastuzumab antibody design 7 (D7), PELLGGPSV (SEQ ID NO: 177) was replaced with LSGRSDNH (SEQ ID NO: 77) two amino acids (PA) after the hinge disulphide bond region (CPPC (SEQ ID NO: 79)). Residues 1.5-4 from the IMGT unique numbering for the C-domain were substituted with a protease sequence, or residues 232-240 from the EU numbering scheme, or residues 245-253 from the Kabat and Chothia numbering schemes.

[0987] Engineered trastuzumab antibody design 7, D7 (SEQ ID NO: 117)

[0988] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKRVEPKSCDKTHTCPPCPALSGRSDNHFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREP QVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR WQQGN VFSCSVM H EALH N HYTQKSLSLSPG K

[0989] Example 2: Molecular Biology

[0990] Engineered trastuzumab geneblock designs 1-3

[0991] Geneblocks of trastuzumab were synthesised (Integrated DNA Technologies) with Engineered trastuzumab geneblock designs 1 to 3 (DI to D3), as given below:

[0992] The final sequences (DNA and protein) for DI - D5 are also shown below. The following applies to SEQ ID NOs 104, 106 and 108:

[0993] • bold underline = protease site (uPA) (SEQ ID NO: 144: CTCTCCGGTCGATCCGACAACCAC) ;

[0994] • bold = Position 5 (IMGT Hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering); and

[0995] • Underline = position 11 (IMGT hinge numbering), 226 (EU numbering scheme) 239 (Kabat and Chothia numbering). Engineered trastuzumab geneblock design 1 - DNA sequence (GENE002) - (SEQ ID NO: 82) attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCT GCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCAC CTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCT GTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTT CGCGCAGAAGATACTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTA TTGGGGGCAGGGCACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGG CTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTG AACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTC AGTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTT ATATCTGCAACGTAAATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTG GGTCTCTCTCCGGTCGATCCGACAACCACq ctg g a a g a g eg g

[0996] >Trastuzumab Design 1 (DI) Full DNA sequence- HCV052 - (SEQ ID NO: 104) GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC ACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAG AGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTC TCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCAGTCAAGTGGCCTT TACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTA AATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTGGGTCTCTCTCCGG TCGATCCGACAACCACGCTAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTG GGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGA GGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGAC GGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTG GTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCA ACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACA GGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTC AAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACA AGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAG AGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA CGCAGAAGAGTCTCTCCCTGTCCCCGGGTAAA

[0997] Engineered trastuzumab geneblock design 2 - DNA sequence (GENE004) (SEQ ID NO: 83) attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCT GCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCAC CTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCT GTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTT CGCGCAGAAGATACTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTA TTGGGGGCAGGGCACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGG CTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTG AACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTC AGTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTT ATATCTGCAACGTAAATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTC

[0998] TCTCCGGTCGATCCGACAACCACq ctggaagagcgg

[0999] >Trastuzumab Design 2 (D2) Full DNA sequence - PLASMID009 - (SEQ ID NO: 106) GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC ACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAG AGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTC

[1000] TCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCAGTCAAGTGGCCTT TACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTA AATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTCTCTCCGGTCGATC CGACAACCACactAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGA CCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC ATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTG GAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCG TCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCC CTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTT CTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACG CCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTG GCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGA GTCTCTCCCTGTCCCCGGGTAAA

[1001] Engineered trastuzumab geneblock design 3 (D3) - DNA sequence (GENE005) (SEQ ID NO: 84) attaagctcttccctggccGAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCT GCGGTTGAGCTGCGCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCAC CTGGCAAGGGGCTTGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCT GTTAAAGGTCGATTCACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTT CGCGCAGAAGATACTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTA TTGGGGGCAGGGCACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGG CTCCAAGCTCTAAGAGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTG AACCTGTGACCGTCTCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTC AGTCAAGTGGCCTTTACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTT ATATCTGCAACGTAAATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTG

[1002] GGTCTCTCTCCGGTCGATCCGACAACCACGGGTCTq ctggaagagcgg

[1003] >Trastuzumab Design 3 (D3) Full DNA sequence - PLASMID010 - (SEQ ID NO: 108) GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA

[1004] CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC ACCCTGGTCACTGTATCCTCTGCCTCTACAAAAGGTCCATCCGTATTCCCATTGGCTCCAAGCTCTAAG AGTACTTCTGGTGGTACAGCCGCCCTTGGATGTCTCGTAAAAGATTACTTCCCTGAACCTGTGACCGTC TCCTGGAATTCAGGGGCACTGACAAGCGGCGTTCATACTTTTCCTGCCGTTCTTCAGTCAAGTGGCCTT TACAGTCTGAGTTCCGTCGTAACAGTCCCTAGCTCAAGTCTTGGGACACAGACTTATATCTGCAACGTA

[1005] AATCACAAACCCTCTAATACTAAGGTAGACAAGAAAGTGGAGCCCAAATCCTGTGGGTCTCTCTCCGG TCGATCCGACAACCACGGGTCTactAGCGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAA CTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTAC GTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTAC

[1006] CGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGG TCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAA CCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCC TGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA CTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGG

[1007] ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCA CTACACGCAGAAGAGTCTCTCCCTGTCCCCGGGTAAA

[1008] All inserts contained the type II restriction enzyme site SapI to enable high throughput cloning.

[1009] Destination vectors were modified with SapI restriction sites to enable VH+ / CH1 domains to be cloned in via a high throughput method. The destination vectors are for mammalian expression of antibodies and include a hinge and an Fc region.

[1010] 100 ng of each geneblock was mixed with 100 ng of pCREA high throughput mammalian Fc vectors along with 5 units of SapI restriction enzyme (New England Biolabs) and 200 units of T4 DNA ligase (New England Biolabs). The samples were left to incubate for 60 minutes at 37°C, then deactivation of the enzyme at 65°C for 20 minutes. The DNA was transformed into NEB 5 alpha cells (New England Biolabs) and plated on LB agar plates containing 100 pg / ml of carbenicillin. Plates were incubated at 37°C overnight. 5 colonies were picked the following day and the correct inserts were checked by mini-prep followed by Sanger sequencing. For large scale preparation of DNA for transfection, the correct constructs were prepped using the endotoxin free maxi prep kit (Qiagen).

[1011] Engineered trastuzumab geneblock designs 4-7 (D4 to D7)

[1012] As designs 4-7 were of closer proximity to the hinge, these constructs were prepared by making geneblocks (Integrated DNA Technologies) that spanned over CHI and CH2 and are as follows.

[1013] The final sequences (DNA and protein) for D4 - D5 are shown below. The following applies for SEQ ID NO 110 and 112 :

[1014] • bold underline = protease site (uPA) (SEQ ID NO: 144: CTCTCCGGTCGATCCGACAACCAC) ;

[1015] • bold = Position 5 (IMGT Hinge numbering), 220 (EU numbering) and 233 (Kabat and Chothia numbering); and

[1016] • Underline = position 11 (IMGT hinge numbering), 226 (EU numbering scheme) 239 (Kabat and Chothia numbering).

[1017] Engineered trastuzumab geneblock (CH I CH2) design 4 (D4) - DNA sequence (GENE006) - (SEQ ID NO: 85) atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC CCAAATCTTGTCTCTCCGGTCGATCCGACAACCACTGCCCACCGTGCCCAGCACCTGAACTCCTGG GGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAG GTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACG GCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGT CAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAAC AAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGG TGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatc

[1018] >Trastuzumab Design 4 (D4) Full DNA sequence - PLASMID014 - (SEQ ID NO: 110) GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC ACCCTGGTCACTGTATCCTCTGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTCTCTCCG GTCGATCCGACAACCACTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCC TCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTG GACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG CCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCT GCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCC ATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATC CCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGAC ATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTG GACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGTCTCTCCCTG TCCCCGGGTAAA

[1019] Engineered trastuzumab geneblock (CH I CH2) design 5 (D5) - DNA sequence (GENE007) - (SEQ ID NO: 86) atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC CCAAATCTTGTCTCTCCGGTCGATCCGACAACCACacaTGCCCACCGTGCCCAGCACCTGAACTCCT GGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTG AGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGA CGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGT GGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCC AACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCAC AGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatctc

[1020] >Trastuzumab Design 5 (D5) DNA sequence - PLASMID011 - (SEQ ID NO: 112) GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC ACCCTGGTCACTGTATCCTCTGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTCTCTCCG GTCGATCCGACAACCACacaTGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTT CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG

[1021] TGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAA

[1022] TGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTC

[1023] CTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCC

[1024] CCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCC

[1025] ATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGC

[1026] GACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTG

[1027] CTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGG

[1028] GGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGTCTCTCC CTGTCCCCGGGTAAA

[1029] The final sequences (DNA and protein) for D6 and D7 are shown below. The following applies for SEQ ID NOs 114 and 116:

[1030] • bold underline = protease site;

[1031] • bold = Position 1.6 (IMGT unique numbering for C-domain), 231 (EU numbering) 244 (Kabat and Chothia numbering; and

[1032] • Underline = Position 5 (IMGT unique numbering for C-domain), 241 (EU numbering) and 255 (Kabat and Chothia numbering).

[1033] Engineered trastuzumab geneblock (CH I CH2) design 6 (D6) - DNA sequence (GENE008) -

[1034] (SEQ ID NO: 87) atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG

[1035] CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC

[1036] ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC

[1037] CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACTCTCCGGTCGATCCGACAACCA

[1038] CCCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCT

[1039] CCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAA

[1040] CTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAG

[1041] CACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAG

[1042] TGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCC

[1043] CCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatc

[1044] >Trastuzumab Design 6 (D6) Full DNA sequence - PLASMID012 - (SEQ ID NO: 114)

[1045] GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC

[1046] GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT

[1047] TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT

[1048] CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA

[1049] CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC

[1050] ACCCTGGTCACTGTATCCTCTGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA

[1051] GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG

[1052] GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC

[1053] AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAAC

[1054] TCACACATGCCCACCGTGCCCAGCACTCTCCGGTCGATCCGACAACCACCCTGAACTCCTGGGGGG

[1055] ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCA

[1056] CATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGT

[1057] GGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGC

[1058] GTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAG

[1059] CCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTA

[1060] CACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGC

[1061] TTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCA

[1062] CGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGG

[1063] TGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAA GAGTCTCTCCCTGTCCCCGGGTAAA

[1064] Engineered trastuzumab geneblock (CH I CH2) design 7 (D7) - DNA sequence (GENE009) -

[1065] (SEQ ID NO: 88) atgccattgtaACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGG

[1066] CTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGC

[1067] ACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGC

[1068] CCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACTCTCCGGTCGATCCGACAACCA

[1069] CTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGG

[1070] TGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCA

[1071] TAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACC

[1072] GTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAG

[1073] CCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCC

[1074] CCCATCCCGGGAGGAGATGACCAAGAACCAGGTattcgcaatc

[1075] >Trastuzumab Design 7 (D7) Full DNA sequence - PLASMID013 - (SEQ ID NO: 116)

[1076] GAGGTTCAACTGGTTGAGAGTGGAGGGGGATTGGTGCAGCCAGGCGGCAGTCTGCGGTTGAGCTGC

[1077] GCAGCCTCTGGATTCAATATCAAAGATACTTACATACACTGGGTCCGACAGGCACCTGGCAAGGGGCT

[1078] TGAGTGGGTTGCCCGTATTTATCCCACTAACGGTTATACACGGTACGCCGACTCTGTTAAAGGTCGATT

[1079] CACAATAAGTGCAGATACCTCCAAAAACACAGCTTATCTGCAAATGAACAGCCTTCGCGCAGAAGATA

[1080] CTGCAGTTTATTATTGCTCACGCTGGGGTGGGGATGGCTTCTACGCTATGGACTATTGGGGGCAGGGC

[1081] ACCCTGGTCACTGTATCCTCTGCTTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCACCCTCCTCCAA

[1082] GAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG

[1083] GTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAG

[1084] GACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGC

[1085] AACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAGAGTTGAGCCCAAATCTTGTGACAAAAC

[1086] TCACACATGCCCACCGTGCCCAGCACTCTCCGGTCGATCCGACAACCACTTCCTCTTCCCCCCAAAA CCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACG AAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCC GCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGG CTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCAT CTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATG ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGT GGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTC CTTCTTCCTCTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCT CCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGTCTCTCCCTGTCCCCGGGTAAA

[1087] The 5' restriction enzyme was Agel and 3' was BsrGI. The destination vector was trastuzumab with Engineered trastuzumab antibody design 1 (SEQ ID NO: 105).

[1088] The geneblocks were cut with Agel-HF (New England Biolabs) and BsrGI (New England Biolabs), as was the vector trastuzumab in the pCREA vector. The samples were left to digest overnight at 37°C. The geneblocks were purified by a PCR purification kit, but the vector was run on a 1% agarose gel in Tris-acetate-EDTA (TAE) buffer to separate the insert from the backbone. The larger backbone fragment was band extracted. Ligations were set up in a 3: 1 molar ratio of insert to backbone of the cut geneblocks and cut backbone. Ligations were with 400 units of T4 ligase (New England Biolabs) and were left overnight at room temperature. Ligations were transformed into NEB 5 alpha cells (New England Biolabs) and spread onto LB agar plates with 100 pg / ml of ampicillin. 5 colonies were checked for the presence of the correct insert by mini prep followed by Sanger sequencing. For large scale preparation of DNA for transfection, the correct constructs were prepped using the endotoxin free maxi prep kit (Qiagen).

[1089] Example 3: Antibody Expression and Purification

[1090] CHO-K1 cell lines

[1091] Trastuzumab in the pCREA antibody mammalian expression vector, alongside the engineered trastuzumab antibody design 1 in the pCREA antibody mammalian expression vector were transfected into CHO-K1 cells by either polyethylenimine (PEI) or expifectamine in a 3: 1 ratio of DNA to PEI only in 30 ml shake flasks. Cells were grown for up to 7 days (96 hours posttransfection), with monitoring of cell health and titre from days 1-4. Comparison of cell health over the first 4 days shows that the engineered trastuzumab antibody design 1 (DI) is indistinguishable from trastuzumab monoclonal antibody. Assessment of antibody concentration by Protein A Octet revealed that there are no differences between the monoclonal antibody and design 1 equivalent for trastuzumab (as shown in Table 1 and Figure 7). At 96 hours post-transfection, expression levels were between 0.891 and 3.02 mg / L. SDS-PAGE was used to look for overexpression, however as expression levels were overall low, this could not be detected (as shown in Figure 8). no Table 1 - Summary of antibody titre

[1092] Transfection conditions were optimised, and cells were grown for up to 7 days, with monitoring of cell health and titre from days 1-4. Again, comparison of cell health over the first 4 days shows that the engineered trastuzumab antibody design 1 is indistinguishable from trastuzumab monoclonal antibody. Assessment of antibody concentration by Protein A Octet revealed that there are no differences between the monoclonal antibody and design 1 equivalent for trastuzumab (as shown in Table 2 and Figure 9). At 96 hours post-transfection, expression levels were between 1.83 and 5.46 mg / L. SDS-PAGE was used to look for overexpression and overexpression of the antibody heavy chains could be detected (see box in Figure 10).

[1093] Table 2 - Summary of antibody titre in optimised experiment

[1094] Expi-CHO cell lines

[1095] After cloning, the trastuzumab in the pCREA antibody mammalian expression vector, alongside engineered trastuzumab antibody designs 1 to 7 (DI to D7) in the pCREA antibody mammalian expression vector, were diluted to a concentration of 1 pg / pl and then transfected into Expi- CHO cell lines (Invitrogen), by Optipro+ ExpiFectamine in shake flasks and incubated at 37°C overnight. The cultures were fed and then the cells were grown for 9 days.

[1096] Purification

[1097] On day 9 the cells were harvested by spinning them down at 3000x g for 30 minutes and filtered through a 0.2 pm filter to recover the overexpressed antibody from the cell supernatants. The supernatants were purified on MabSelect PrismA VI ml prepacked columns using the AKTA Go Instrument (Cytiva). Elution was carried out at pH 2.7 in HiTrap® MabSelect Binding Buffer (NaP04) and HiTrap® MabSelect Elution Buffer (Glycine). The relevant fractions were pooled according to chromatograms and the buffer exchanged in lx DPBS using Vivaspin20 concentrators at 3800x g for 20 minutes (as shown in Figure 11 for DI). The absorbance was measured using a Nanodrop (ThermoScientific), and the yield and concentration was calculated. Trastuzumab and engineered antibodies Dl-7 have similar expression levels and yields (as shown in Table 3).

[1098] Table 3 - Summary of antibody expression level and yield SDS-paqe analysis of purified control and D1-D7

[1099] The purified antibodies were heated at 85°C for 5 minutes, then 10 pl of which were loaded onto a NuPAGE 4-12% Bis-Tris Gel inserted in a mini Gel Tank filled with IX MES SDS running buffer, with a protein marker loaded as molecular weight standard. The gel was run for 25 minutes at 200 V, then stained with InstantBlue® protein stain and subsequently destained with H2O.

[1100] Both the reduced and non-reduced SDS-PAGE gels showed mAb-like antibody profiles (as shown in Figure 12). All molecules appeared as expected, with D5 being the biggest variant as it has a higher molecular weight due to the engineered glycosylation site. These results are consistent with the results observed in the size exclusion (SEC) high-performance liquid chromatography (HPLC) assays and Tmand Taggdetermination assays analysis below.

[1101] SEC-HPLC Assay of purified engineered antibodies

[1102] 10 pg of the purified antibody samples were injected into a MAbPac SEC-1 (4x300 mm) column with precolumn MabPac SEC-1 (4x50mm) and a SECHPLC assay was performed to determine the percentage monomer to higher order oligomers present.

[1103] The SEC-HPLC assay demonstrated good solution state profiles of Dl-7 (as shown in Table 4) showing that they all had acceptable levels of purity comparative to the control, trastuzumab.

[1104] Table 4 - Thermal Stability Analysis of mAb Control and Dl-7

[1105] Thermal stability of purified engineered antibodies

[1106] The samples were diluted to 1 mg / mL in DPBS (Dulbecco's Phosphate Buffered Saline), then 9 pL of each sample was loaded in triplicate into the UNCLE instrument (Unchained Labs) and the melting temperature (Tm) and aggregation temperature (Tagg) were determined.

[1107] All antibodies showed high TM and Taggvalues (Table 4). All antibodies presented TM values above 65.0°C, therefore meeting the usual requirement for CMC development, except D7 which showed the lowest TM of 47.4°C (as shown in Table 4). D7 was also the only sample to exhibit a second TM (76.1 °C). D4 presented the highest TMI value (70.8 °C), while D7 showed the lowest (47.4 °C). The highest Taggwas observed for D5 (74.5 °C), and the lowest for the trastuzumab control (67.1 °C).

[1108] Conclusion

[1109] Considering the analysed biophysical parameters, engineered D1-D7 have been shown to maintain the biophysical and biochemical properties required of trastuzumab for functioning as an antibody and such properties have not been negatively impacted by the engineering of adding the cleavable site.

[1110] Example 4: Antibody characterisation

[1111] Binding of purified engineered antibodies to hHER2 (assessed by ELISA)

[1112] Maxisorp™ ELISA plates were prepared by adding 50 pl of either phosphate buffered saline (PBS), bovine serum albumin (BSA - lug / ml) or hHER2 (1 ug / ml) to each well and placed in the fridge overnight. The plates were washed three times with PBS+0.1% Tween20 and three times in PBS and left to block with 200 pl of PBS+6% dried milk for one hour at room temperature before being washed again.

[1113] The purified trastuzumab mAb and Dl-7 antibodies were diluted to 50 pl per well in PBS+3% dried milk, then added to the plates and incubated for 1 hour at room temperature, after which the plates were washed again. Antibodies were added at a top concentration of 150-50 nM and diluted 1 in 2, or 1 in 3 over an 8 or 12 point dilution series. 50 pl of a 1 in 5000 dilution in PBS+3% dried milk of the anti Vk antibody-HRP conjugated was added to each well, then left to bind to the samples at room temperature for 1 hour. The plates were washed again, and 50 pl of 3,3',5,5'-Tetramethylbenzidine (TMB) peroxidase substrate was added to develop the colour. The signal was quenched with 50 pl of IM HCI and the plates were read for absorbance at 450 nm.

[1114] D1-D7 show identical binding to hHER2 by ELISA (as shown in Table 5 and Figure 13).

[1115] Table 5 - IC50 values of trastuzumab control and D1-D7

[1116] Binding of purified engineered antibodies to hHER2 (assessed by BIAcore)

[1117] A CM5 chip was coated with anti-human IgG with 12,500 RU units, using conditions of Cytiva® HBS-EP (0.01 M HEPES pH 7.4, 0.15 M NaCI, 3 mM EDTA, 0.005% v / v Surfactant P20) + running buffer and regeneration with 0.1 M glycine pH 2.0. The hHER2 protein did not show binding to the blank CM5 chip.

[1118] Anti-Human IgG Fc antibody was then immobilised to 10,215.8 RU units on flow cell (Fc) 2 of a CM5 chip using amine coupling chemistry, using HBS-EP+ running buffer. hHER2 protein was then shown not to bind to the immobilised anti-human IgG Fc antibody.

[1119] Capture optimisation of trastuzumab (at 0.1, 0.5 and 1 pg / ml) using hHER2 (at 1, 10 and 100 nM) was performed. Trastuzumab showed stable capture levels and capture concentrations, and its capture baseline did not vary much between cycles which implied efficient regeneration using 0.1M glycine HCI pH 2.0.

[1120] A 12-point dilution kinetics measurement of trastuzumab and D1-D7 was conducted using HBS- EP + running buffer with regeneration with 0.1 M glycine bound to hHER2-HIS with a similar KD and the samples were assayed at room temperature. All D1-D7 showed identical binding to hHER2 by BIAcore (as shown in Table 6 and Figure 14). This shows that the engineered cleavable domain has not reduced or otherwise negatively impacted the antibody's ability to bind to hHER2.

[1121] Table 6 - Binding of Dl-7 and control to hHER2 Protease assay of purified antibodies

[1122] Conditions for the protease assay were optimised and protease buffer (50 nM Tris-HCI and 0.01% Tween-20 pH 8.0) was used to dilute the purified Dl-7 antibodies and control trastuzumab. Each sample was digested with 0 nM, 23.5 nM and 235 nM of uPA enzyme and incubated at 37°C for 24 hours. 5 pl of NuPAGE™ LDS Sample Buffer (Thermo Fisher Scientific®) loading dye with 10 nM of dithiothreitol (DDT) was added to each sample and the samples were boiled at 95°C for 10 minutes, then cooled to 10°C in a PCR machine. The entire contents were loaded onto a 4-12% SDS PAGE gel and run for 30 minutes at 200°C. The gel was left to stain for a few hours at room temperature in InstantBlue® stain, then left to destain in water over a few days.

[1123] It was found that the uPA protease liberates Fab or F(ab')2 fragments from D1-D4 and D6-D7 (as shown in Figure 15). The uPA enzyme did not digest the trastuzumab control. This experiment showed that the engineered D1-D4 and D6-D7 with protease cleavage sites were successfully cleaved, whereas the controls, D5 and trastuzumab, without protease cleavage sites, did not cleave.

[1124] Binding of uPA-treated cleaved engineered antibodies to hHER2 (assessed bv ELISA)

[1125] 4 ELISA plates were set up with hHER2-HIS (50 pl) at 1 pg / ml in PBS and 2 ELISA plates were set up with PBS (50 pl) to test the binding of the cleaved therapeutic domain to its binding target. The plates were covered and left at 4°C overnight.

[1126] The plates were removed from the fridge and washed (as described above), then serially diluted 1 in 4. Each antibody was assayed in triplicate, one set each for the PBS plate, hHER2-HIS plate (Vk detection) and hHER2-HIS plate (Fc detection). The antibodies were left to incubate for 1 hour at room temperature. The plates were washed again, then the antibodies were added to the plates. The anti-Vk antibody was diluted as described above and added to 1 set of hHER2- HIS plates and the PBS plates. 50 pl was added to each well. The plates were left to incubate for 1 hour at room temperature, then washed again. The plate was developed, then the signal was quenched, and the plate was read for its absorbance at 450 nm.

[1127] It was found that uPA treatment does not affect antibody binding to hHER2 (as shown in Table 7 and Figure 16).

[1128] Table 7 - IC50 values of uPA-treated trastuzumab control and D1-D7

[1129] Binding of uPA-treated cleaved engineered antibodies to Fc gamma receptors (assessed by ELISA)

[1130] ELISA plates were made up to test the binding of the stabilisation domain to Fc gamma receptors (I, Ila, lib, Illa). Fifty pl of 1 pg / ml of Fc gamma RI (CD64), Fc gamma Rlla (CD32a), Fc gamma Rllb (CD32b), Fc gamma RHIa (CD16a V176) and 50 pl of PBS were added to each well. The plates left at 4°C overnight. The plates were washed, left at room temperature for 1 hour to block, then washed again.

[1131] The antibodies were tested in triplicate over an 8-point dilution with a 1 in 3 dilution factor. Fifty pl of PBS+3% dried milk was added to each well. The samples were left for 1 hour at room temperature for binding and the plates were washed. Anti-kappa IgG was diluted 1 in 5000 in PBS+3% dried milk. Samples were left to incubate for 1 hour at room temperature and the plates were washed again. The plate was developed, then the signal was quenched, and the plate was read for its absorbance at 450 nm.

[1132] It was found that the engineered antibodies do not affect Fc gamma receptor la and Ila binding (as shown in Figures 17A and 17B). All of D1-D7 bound to Fc gamma receptor la strongly. D7 binding to Fc gamma receptor la was impacted by the protease site, which is expected to be due to the crystal structure of how the Fc region interacts with the Fc gamma receptors. There was no observed binding to Fc gamma receptor lib (as shown in Figure 17C). Binding to Fc gamma receptor Illa was assessed using the 176V variant; both D6 and D7 showed a slightly decreased binding, whereas D5 showed increased binding (Figure 17D) which is thought to be due to the glycosylation. All other engineered trastuzumab variants (D1-D4) showed similar binding to Fc gamma receptor Illa as the trastuzumab mAb control.

[1133] Conclusion

[1134] The engineering of a cleavable domain into an antibody-derived fragment has minimal effects of the ability of the stabilisation domain to bind to Fc gamma receptors. Example 5: Manufacturability of engineered agents

[1135] Transfection of Trastuzumab and D3

[1136] Trastuzumab and D3 in pCREA antibody mammalian expression vectors were diluted to a concentration of 1 pg / pl and then the transfected into Expi-CHO cell lines (Invitrogen), by Optipro+ ExpiFectamine in shake flasks and incubated at 37°C overnight. The cultures were fed and then the cells were grown for 10 days.

[1137] On day 10, the cultures were split in 500 ml conical tubes and spun at 3900x g for 30 minutes. The supernatants were then filtered through a 20 pm filter. 30 ml was transferred to a 50 ml Falcon tube and filtered, and 250 ml was purified to obtain large scale purified preparations.

[1138] Purification of Trastuzumab and D3

[1139] The harvested trastuzumab and D3 were purified on MabSelect PrismA™ 1 mL columns using the AKTA Go Instrument (Cytiva). The relevant fractions were pooled according to chromatograms and the buffer exchanged in lx DPBS using Vivaspin20 concentrators at 3800x g for 20 minutes. The absorbance was measured using a Nanodrop (ThermoScientific), and the concentration was determined (as shown in Table 8).

[1140] Table 8 - Concentration of Trastuzumab and D3 samples after Protein A purification

[1141] Quality Control (QO analyses of Trastuzumab and D3

[1142] 1 mg / ml of each sample was analysed on an isocratic run at 0.35 ml / min for 15 minutes, using a Dionex UltiMate 3000 UHPLC system (Thermo Scientific). SEC-HPLC analysis of trastuzumab showed a 98.5% monomer purity and D3 a 94.3% monomer purity (as shown in Figure 18).

[1143] 1.5 pg of each sample was analysed on a 4-12% NuPAGE SDS gel and showed good purity of D3 (as shown in Figure 19). The samples were filtered and aliquoted into 2 ml tubes (as shown in Table 9).

[1144] Table 9 - Final Concentration of Trastuzumab and D3 samples after filtration Binding of large-scale preparations of Trastuzumab and D3 to hHER2 (assessed by ELISA)

[1145] An ELISA plate was prepared with 1 pg / ml of hHER2-HIS diluted in PBS, a second ELISA plate was prepared with 1 pg / ml of BSA and a third ELISA plate was prepared with PBS alone. The plates were left in the fridge overnight. The plates were then washed, left for 1 hour at room temperature to block and then washed again, as described in Example 4.

[1146] 150 pl of the large-scale purified preparations (trastuzumab and D3 antibodies) were transferred into 150 pl of PBS+3% dried milk, over a 12-point dilution. 50 pl was added to each well and incubated for 1 hour at room temperature. The plates were washed, the colour was developed and the signal quenched as described above. The plate was read for absorbance at 450 nm.

[1147] Trastuzumab and D3 from large-scale expression preparations retained their ability to bind to hHER2. Both trastuzumab and D3 were found to be active. Trastuzumab bound hHER2 with an IC50 of 0.6095 nM and D3 bound hHER2 with an IC50 of 0.1035 nM (as shown in Table 10 and Figure 20).

[1148] Table 10 - IC50 values of Trastuzumab and D3 from ELISA

[1149] Purification of trastuzumab and DI

[1150] The trastuzumab control and DI were expressed from the transfected CHO-K1 cells, as described in Example 3. The samples were spun down at 3900x g for 20 minutes and filtered through a 0.22 pm filter, then loaded onto a MabSelect PrismA 1 ml prepacked column and purified using the AKTA Go Instrument (Cytiva). The relevant fractions were pooled, and buffer was exchanged in DPBS using a Vivaspin20, 30 kDa MWCO and spinning in a benchtop centrifuge at 3800x g for 20 minutes. The absorbance was measured using a Nanodrop (ThermoScientific), and the yield and concentration was calculated. It was found that expression levels for both trastuzumab and DI were very low, likely due to a plasmid / cell line lack of optimisation (as shown in Table 11). However, the expression levels between the two were comparable showing the engineered agent maintains the purity of trastuzumab.

[1151] Table 11 - Expression levels of CHO-K1 Trastuzumab and DI SDS-PAGE analysis of purified trastuzumab mAb and DI

[1152] The purified trastuzumab and DI samples were boiled at 85°C for 5 minutes, then separated by SDS-PAGE. The gel showed intact antibody for both trastuzumab and DI (as shown in Figure 21).

[1153] Binding of Trastuzumab and DI expressed from Expi-CHO and CHO-K1 cells to hHER2 (assessed bv ELISA)

[1154] Three Maxisorb ELISA plates for each antigen were prepared by adding either 50 pl of PBS, 1 pg / ml of BSA or 1 pg / ml of hHER2 in each well and placed into the fridge overnight. The plates were washed and left to block at room temperature for 1 hour, then washed again. The antibody samples were purified according to Examples 3 and 6 above, were diluted 1 in 3 to a final volume of 50 pl per well, and each antibody was assayed in triplicate over an 8-point dilution. The samples were diluted in PBS+3% dried milk and incubated for 1 hour at room temperature. The plates were prepared with the anti-Vk antibody as described above. The samples were left to bind at room temperature for 1 hour. The plates were washed as described above. The colour was developed, the signal quenched and the plate was read for absorbance at 450 nm.

[1155] Trastuzumab and DI expressed from Expi-CHO or CHO-K1 cells were identical as judged by ELISA and the potency of trastuzumab was not compromised by insertion of an exogenous cleavable domain (as shown in Figure 22 and Table 12).

[1156] Table 12 - IC50 values of expression of trastuzumab and DI from CHO-K1 and Expi-CHO

[1157] Example 6: Different Protease Cleavage Sites are amenable to the engineered agent design

[1158] To demonstrate that this technology can be used with different types of cancer specific protease cleavage sites, different agents were designed with an ADAM 10 site (D9). This protease was selected because it belongs to different protease family than uPA (which is a serine proteases). ADAM10 is a metalloprotease Napsin-A. The designs were based on the engineered trastuzumab antibody design 3. Cloning

[1159] The constructs incorporating the different protease cleavage site (ADAM 10 site: PRAEALKGG (SEQ ID NO: 89, underlined) was designed and expressed. The constructs were based on the engineered trastuzumab antibody design 3.

[1160] Engineered antibody with ADAM 10 site

[1161] In the engineered trastuzumab antibody design 9 (D9) GSPRAEALKGGGSAS (SEQ ID NO: 90) was added after the cysteine residue that ends the CHI domain and prior to the DKTHT (SEQ ID NO: 78) hinge region (italicised). This adds an additional 2 amino acids on the C terminal end of the protease cleavage site and an additional 4 amino acids on the N terminal end of the protease cleavage site for greater accessibility.

[1162] Engineered trastuzumab antibody design 9, D9 (SEQ ID NO: 8) :

[1163] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKP SNTKVDKKVEPKSCGSPRAEALKGGGSASDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLD SDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[1164] A geneblock of D9 (SEQ ID NO: 91) was synthesised (Integrated DNA T...

Claims

CLAIMS1. A pharmaceutical composition comprising an engineered agent and at least one pharmaceutically acceptable carrier, diluent or excipient, wherein the agent comprises: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain, wherein the cleavable domain comprises a tumour-specific protease cleavage site and is positioned between the therapeutic domain and the stabilisation domain, and wherein the pharmaceutically acceptable carrier, diluent or excipient is the pharmaceutically acceptable carrier, diluent or excipient is a buffer, a sugar, an isotonic agent, an antioxidant, an amino acid, a chelator, a surfactant, an emulsifier and / or saline.

2. A pharmaceutical composition comprising an engineered agent and at least one pharmaceutically acceptable carrier, diluent or excipient, wherein the agent comprises: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilisation domain, and wherein the pharmaceutically acceptable carrier, diluent or excipient is selected from any one or more of citric acid, dextrose, acetic acid, glutamic acid, glycine, L-histidine, L-histidine monohydrochloride monohydrate, L-lysine, L-lysine hydrochloride, L-methionine, L- phenylalanine, L-threonine, mannitol, monosodium glutamate, pentetic acid, polysorbate 20, polysorbate 80, sodium acetate trihydrate, sodium chloride, sodium citrate, sorbitol, sucrose, trehalose, maltose, ethylenediaminetetraacetic Acid (EDTA), Tween, glycerol, a glycol, ethanol, fructose, glycerine, glucose and / or sucralose.

3. A medical container comprising an engineered agent, wherein the agent comprises: i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain, wherein the cleavable domain is positioned between the therapeutic domain and the stabilisation domain.

4. A medical container comprising a pharmaceutical composition according to Claim 1 or 2.

5. The medical container comprising according to Claim 3 or 4, wherein the medical container is a vial, syringe, bag or injection device, optionally wherein the bag is an intravenous (IV) bag, optionally wherein the bag, syringe or injection device is for intravenous,subcutaneous, intramuscular, intradermal and / or intraosseous delivery, preferably wherein the bag, syringe or injection device is for intravenous or subcutaneous delivery.

6. A kit comprising:(a) an engineered agent, and(b) a label or instructions for use, wherein the agent comprises i. a therapeutic domain; ii. a cleavable domain; and iii. a stabilisation domain, and wherein the cleavable domain is positioned between the therapeutic domain and the stabilisation domain.

7. A kit comprising:(i) a pharmaceutical composition according to Claim 1 or 2; and(ii) a label or instructions for use.

8. The kit according to Claim 6 or 7, wherein the label or instructions comprise a marketing authorisation number (e.g., an FDA or EMA authorisation number).

9. The kit according to any one of Claims 6 to 8, wherein the instructions indicate that the agent or pharmaceutical composition is for use to treat and / or prevent a tumour in a human.

10. The kit according to any one of Claims 6 to 8, wherein the instructions indicate that the agent or pharmaceutical composition is for use to treat and / or prevent a cancer in a human, preferably wherein the cancer is a solid tumour.

11. An antibody-drug-conjugate (ADC) comprising: i. a therapeutic domain; ii. a cleavable domain; iii. a stabilisation domain; and iv. one or more payloads; wherein the cleavable domain is positioned between the therapeutic domain and the stabilisation domain.

12. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the therapeutic domain is: i. an antigen-binding domain; ii. a Fab region;iii. a F(ab')2 region; iv. a scFv region; v. a tandem scFv region; and / or vi. a sdAb.

13. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the agent is a monoclonal antibody.

14. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the stabilisation domain is a Fc region, optionally wherein the Fc region is an IgG, IgE, IgM, IgD or IgA family Fc region or a bispecific Fc region.

15. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the protease is a protease that is expressed by and / or accumulates in the vicinity of unwanted cells or a tumour.

16. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the protease is a tumour-specific protease such as u plasminogen activator (uPA), Napsin A, ADAM10, FAP, matriptase, legumain, MT-SP1, cysteine proteases, serine proteases, metalloproteases, or combination thereof.

17. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the cleavable domain is located in a hinge region, optionally wherein the cleavable domain is located in the upper hinge or in the lower hinge.

18. The pharmaceutical composition, kit, medical container or ADC of any one of the preceding claims, wherein the agent is a protein or polypeptide, the stabilisation domain comprises a Fc region and a hinge region and the cleavable domain is located i. N-terminal to the hinge region and after the therapeutic domain; or ii. C-terminal to the hinge region and before the CH2 domain of the Fc region.

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