WO2026038048A2 - Agents, procédés et utilisations de ceux-ci - Google Patents

Agents, procédés et utilisations de ceux-ci

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Publication number
WO2026038048A2
WO2026038048A2 PCT/GB2025/051807 GB2025051807W WO2026038048A2 WO 2026038048 A2 WO2026038048 A2 WO 2026038048A2 GB 2025051807 W GB2025051807 W GB 2025051807W WO 2026038048 A2 WO2026038048 A2 WO 2026038048A2
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WIPO (PCT)
Prior art keywords
domain
numbering
agent
cleavable
therapeutic
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Pending
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PCT/GB2025/051807
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WO2026038048A3 (fr
Inventor
Zahra JAWAD
Joyce RATTI
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Creasallis Ltd
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Creasallis Ltd
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Publication of WO2026038048A2 publication Critical patent/WO2026038048A2/fr
Publication of WO2026038048A3 publication Critical patent/WO2026038048A3/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C07K2317/53—Hinge
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/55—Fab or Fab'
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/64—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising a combination of variable region and constant region components
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site

Definitions

  • the present invention relates to methods and conjugates to improve the penetrability of biological substances into the tumour microenvironment (TME) for therapeutic purposes.
  • 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.
  • 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.
  • antibody-based therapies are 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).
  • Antibodies also contain a fragment crystallisable (Fc) region.
  • Fc fragment crystallisable 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.
  • GFB glomerular filtration barrier
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • FIG. 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).
  • FIG 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.
  • 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.
  • conditions allow for the cleavage of the agent into 2 domains, the therapeutic domain (xl) and Fc domain (xl).
  • 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.
  • Figure 6 Illustrative schematic of different positions of the cleavable domain within the hinge region.
  • D1-D4 designs - the cleavable site is within the upper hinge region;
  • D6-D7 designs - the cleavable site is within the lower hinge region;
  • 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).
  • Figure 8 Gel image showing Protein Quality Analysis (SDS-PAGE).
  • FIG 10 Repeat gel image showing Protein Quality Analysis (SDS-PAGE). The box shows the antibody heavy chains ( ⁇ 50kDa).
  • Figure 12 Gel image showing (A) Non-Reduced Protein Quality Analysis (SDS-PAGE) and (B) Reduced Protein Quality Analysis (SDS-PAGE).
  • Figure 13 Binding of trastuzumab mAb and engineered designs to hHER2 (human HER2).
  • Fc is the middle row of bands
  • 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.
  • FIG 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.
  • Figure 18 Larger Scale 20mg Expression of (A) Trastuzumab mAb 98.5% monomer and (B) Trastuzumab D3 94.3% monomer.
  • FIG 19 SDS PAGE gel showing expression and purity of Trastuzumab-mAb and D3.
  • a PageRuler Plus prestained ladder was used.
  • Figure 20 Binding of Larger Scale Preps of trastuzumab mAb and D3 to hHER2 by ELISA.
  • FIG 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)
  • 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.
  • Figure 26 (A) Binding of Cetuximab mAb and Cetuximab D12 to hEGFR; (B) Binding of Bevacizumab mAb and Bevacizumab D13 to hVEGF.
  • 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.
  • 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.
  • 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.
  • the cleavable domain is located in the upper hinge and the stabilisation domain is an Fc region.
  • Figure 30 SDS PAGE gel showing (A) expression and purity of various engineered agents comprising VHH or ScFv as a therapeutic domain.
  • the agent may be a therapeutic agent.
  • 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.
  • 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.
  • the agent is an engineered agent.
  • engineered agent we include the meaning that the agent is not a naturally occurring agent and may be synthesised.
  • 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.
  • '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.
  • 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.
  • the cleavable domain is a peptide that includes a substrate for an enzyme. In one embodiment, the cleavable domain comprises only the cleavage site.
  • 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.
  • any overlap is preferably at the C terminus of the therapeutic domain and/or at the N-terminus of the stabilisation domain.
  • the agent of the invention does not include a naturally occurring cleavage site.
  • 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.
  • the cleavable domain is artificially introduced into a naturally occurring agent.
  • the agent is engineered such that a cleavable domain is artificially introduced.
  • the cleavable domain is an engineered cleavable domain. In one embodiment, part or all of the cleavable domain has been engineered into an antibody.
  • 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.
  • 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.
  • domain antibodies dAbs
  • diabodies nanobodies (such as camelid antibodies, engineered camelid antibodies, shark antibodies or llama antibodies).
  • 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.
  • 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.
  • 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.
  • 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.
  • '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.
  • the agent comprises amino acids.
  • the agent is a protein, peptide, bicyclic peptide, tricyclic peptide or a polypeptide.
  • 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.
  • the agent comprises non-natural isomers or amino acids.
  • 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.
  • nucleic acid is expressed in a suitable host to produce an engineered agent of the invention.
  • 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.
  • 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.
  • amino acid analogues such as penicillamine, 3-mercapto-D-valine
  • non- proteogenic amino acids such as norleucine
  • beta-amino acids such as norleucine
  • beta-amino acids such as norleucine
  • azapeptides such as azapeptides
  • N-methylated amino acids such as N-methylated amino acids
  • chemically synthesised compounds that have properties known in the art to be characteristic of an amino acid.
  • proteogenic indicates that the amino acid can be incorporated into a protein in a cell through well-known metabolic pathways.
  • the definition also includes
  • 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.
  • 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.
  • Non protein entities 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.
  • the glomerular filtration barrier 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • DLS Dynamic Light Scattering
  • 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
  • the molecular weight of an agent substance 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).
  • 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.
  • 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%.
  • PK pharmacokinetic
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the solid tumour is malignant.
  • the solid tumour can be a carcinoma, a sarcoma, a lymphoma, or a melanoma.
  • 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.
  • 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.
  • 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.
  • %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.
  • the increased penetrability and/or tumour uptake levels are sustained. In another embodiment, the increased penetrability and/or tumour uptake levels are transient.
  • sustained we include the meaning that the increased levels of the agent are maintained over time and/or continue to penetrate the solid tumour.
  • tained 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.
  • 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.
  • the tumour uptake level of the therapeutic domain is increased relative to the tumour uptake level of the agent.
  • 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.
  • 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.
  • 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.
  • the therapeutic domain is not a single domain antibody (sdAb).
  • 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.
  • the therapeutic domain comprises means for binding an antigen, for example any of the antigens described elsewhere herein.
  • the therapeutic domain is or is derived from a Fab region, a F(ab')2 region or a scFv region.
  • 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.
  • the therapeutic region further comprises a hinge region.
  • 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.
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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).
  • 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).
  • N-terminal 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • the two chains are connected with a short linker peptide.
  • 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.
  • the flexible linker allows for the self-assembly of a heavy and light chain variable region.
  • the linker length is a minimal distance of 3.5 nm and/or is at least 12 amino acids.
  • the linker has a maximum length of 30 amino acids.
  • the linker is 12-30 amino acids long.
  • the linker is 12-25 amino acids long.
  • the linker is 15-20 amino acids long.
  • 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.
  • 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.
  • 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.
  • the scFvs are expressed as domains without a N and/or C-terminal appendage, in particular without any purification and/or detection tag.
  • Antibody drug conjugates are molecules comprising an antibody linked to a biologically active cytotoxic drug.
  • the antibody is specific to antigens expressed on the unwanted cells and guides the cytotoxic drug to the required location in the body.
  • 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).
  • a single-domain antibody 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.
  • VHH or VHH fragments from camelids such as llamas, VNAR fragments from cartilaginous fish
  • VL domains or human domain antibodies.
  • 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.
  • 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).
  • the heavy chain is 85, 90 or 95% of the length of the chain described above.
  • 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).
  • the light chain is 85, 90 or 95% of the length of the chain described above.
  • the heavy chain comprises three complementarity determining regions (CDRs):
  • HCDR3 at positions 105-115 (IMGT), positions H95-102 (Kabat), and/or positions H96-101, for CDRH3.
  • the light chain comprises three complementarity determining regions (CDRs):
  • 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.
  • 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.
  • 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.
  • 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).
  • suitable methods for removing purification tags or detection tags e.g. enzymatic cleavage).
  • a monoclonal antibody 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).
  • polyclonal antibodies bind to multiple epitopes (multispecific) and are usually made by several different antibody-secreting plasma cell lineages.
  • the monoclonal antibodies can be engineered to be multispecific in order to increase the number of epitopes that it binds to.
  • the therapeutic domain may be monospecific or multispecific, for example bispecific or trispecific.
  • 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.
  • 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).
  • 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 k
  • 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.
  • 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.
  • 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-CE
  • an antibody-derived fragment may have the same of similar sequences as parts of the parent antibody.
  • 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.
  • an antibody-derived fragment has at least 80%, 82%, 85%, 87% or 89% sequence identity to a part of the parental antibody sequence.
  • an antibody-derived fragment has at least 90%, 92%, 95%, 97% or 99% sequence identity to a part of the parental antibody sequence.
  • an antibody derived fragment is identical to the corresponding part of the parental antibody.
  • 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:
  • Dlx Engineered trastuzumab antibody design lx (SEQ ID NO: 1)
  • D2x Engineered trastuzumab antibody design 2x (SEQ ID NO: 2)
  • D3x Engineered trastuzumab antibody design 3x (SEQ ID NO: 3)
  • D4x Engineered trastuzumab antibody design 4x (SEQ ID NO: 4)
  • D5x Engineered trastuzumab antibody design 5x (SEQ ID NO: 5)
  • D6x Engineered trastuzumab antibody design 6x (SEQ ID NO: 6)
  • LSGRSDNH SEQ ID NO: 77
  • PAP three amino acids
  • CPPC hinge disulphide bond region
  • 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.
  • D7x Engineered trastuzumab antibody design 7x (SEQ ID NO: 7)
  • 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.
  • 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:
  • 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.
  • 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:
  • 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.
  • 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:
  • D14b Engineered pembrolizumab antibody design 14b (SEQ ID NO: 14) :
  • 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.
  • 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) :
  • 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.
  • 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:
  • 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.
  • 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:
  • 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.
  • 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:
  • 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.
  • 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:
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the therapeutic domain is not multivalent.
  • the therapeutic domain targets or guides the agent to unwanted cells.
  • 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.
  • 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.
  • the expressed entity is expressed selectively on the unwanted cell.
  • 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.
  • 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.
  • entity e.g. target and/or antigen
  • 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.
  • entity e.g. target and/or antigen
  • 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 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.
  • affinity 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) .
  • affinity can be determined by any method known in the art and described herein. Any KD value greater than IxlO' 6 M is generally considered to indicate non-specific binding.
  • 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).
  • 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.
  • the binding partner may bind to a tumour associated antigen which is expressed on the cell membrane.
  • the binding partner may bind an entity that is similarly or under-expressed on unwanted cells relative to other cells within the body.
  • the entity is naturally occurring or endogenously expressed.
  • 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).
  • Unwanted cells include cells whose presence in a host or patient is undesired, such as tumour cells or other disease-causing cells.
  • 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.
  • 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.
  • 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.
  • the therapeutic domain does not target the agent to the unwanted cells.
  • 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).
  • Insulin like growth factors are preferentially taken up by malignant cells and so may be used to target tumour cells.
  • EGF can be used to target malignant cells which upregulate the EGF receptor.
  • 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.
  • IL-6 may be used as a therapeutic domain for myeloma.
  • the therapeutic domain is melanoma stimulating hormone (MSH) which binds to the MSH receptor which is expressed in high numbers in melanoma cells.
  • MSH melanoma stimulating hormone
  • 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) CD
  • 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 NO:
  • 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:
  • 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:
  • 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:
  • 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:
  • 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:
  • 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:
  • 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:
  • Engineered PEGylated trastuzumab scFv design 20 (SEQ ID NO: 100) EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTI SADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGGGGSGGGGSD IQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDF TLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK
  • the cleavable domain does not (significantly) interfere with the binding of the agent to its binding partner.
  • 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.
  • a cleaved state therapeutic domain
  • uncleaved state agent
  • 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.
  • 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.
  • 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.
  • the agent does not comprise a masking domain.
  • 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.
  • 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.
  • IC50 half maximal inhibitory concentration
  • 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.
  • ELISA enzyme- linked immunosorbent assay
  • 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,
  • KD dissociation constant
  • 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).
  • 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.
  • 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.
  • a cleaved state therapeutic domain
  • uncleaved state agent
  • 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).
  • the agent has a melting temperature (T m ) 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,
  • the Tm of a protein relates to the result of denaturation of the protein.
  • Methods of measuring melting temperatures include differential scanning calorimetry (DSC), differential scanning fluorometry (DSF) and other well-known thermal shift assays.
  • the melting temperature is measured with an in vitro DSC assay.
  • the agent has an aggregation temperature (T agg ) 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,
  • T agg 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 T agg is measured with an in vitro DLS assay.
  • DLS measures the hydrodynamic size and size distribution of particles in solution and can be plotted over time and temperature.
  • a protein may be stable and show repeatable size (and scattering intensity) measurements, whereas typically at more elevated temperatures (T agg ), protein molecules will show a tendency to aggregate.
  • 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.
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • ADCP antibody-dependent cell-mediated phagocytosis
  • CDC complement-dependent cytotoxicity
  • IgG and neonatal FcR (FcRn) can protect antibodies from being degraded, thereby prolonging its half-life.
  • the cleavable domain may interfere with the binding of the stabilisation domain to Fc gamma receptors.
  • 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.
  • 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.
  • glycosylation is the addition of carbohydrate chains (glycans) proteins.
  • Glycosylation may be O-glycosylation or N-glycosylation.
  • the glycosylation site in the cleavable domain is an N-glycosylation motif.
  • 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.
  • 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.
  • cleaved state stability domain
  • uncleaved state agent
  • Fc gamma receptors Fc gamma receptors
  • the therapeutic domain reduces or inhibits the proliferation of unwanted cells or the growth of a tumour.
  • the therapeutic domain (for example when bound to its target or binding partner) stimulates immune cells, for example immune checkpoint inhibitors (ICIs), immunomodulators, cytokines.
  • immune cells for example immune checkpoint inhibitors (ICIs), immunomodulators, cytokines.
  • 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).
  • 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.
  • the therapeutic domain blocks immune cells.
  • blocks and “inhibits” are used interchangeably and encompass both partial and complete inhibition/blocking.
  • 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.
  • the therapeutic domain may have a secondary function wherein the therapeutic domain targets (e.g. localises) the agent to the target tissue.
  • the therapeutic domain 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.
  • the stabilisation domain is a protein-based domain or a polymer.
  • 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).
  • the stabilisation domain is not a protein, for example the stabilisation domain is a chemical moiety (e.g. PEGylation or hyaluronic acid).
  • the stabilisation domain targets the therapeutic domain to unwanted cells.
  • 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.
  • 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.
  • HSA Human Serum Albumin
  • 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.
  • 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.
  • subtypes of IgG include IgGl, IgG2, IgG3, IgG4
  • 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.
  • the agent is based on an antibody which is human or humanised.
  • 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.
  • 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.
  • the heavy chain constant region e.g. an Fc domain of the stabilisation domain
  • the heavy chain constant region e.g. an Fc domain of the stabilisation domain
  • the heavy chain constant region is IgG4-PE.
  • 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).
  • 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.
  • 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.
  • 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).
  • 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.
  • 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).
  • 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
  • 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.
  • 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
  • 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.
  • 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).
  • 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.
  • the serine at position 10 is substituted for a proline in order to improve structural rigidity.
  • 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).
  • 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):
  • T366W + T366S/L368A/Y407V mutations [preferably in IgGl Fc regions];
  • N297A mutations reduce Fc gamma receptor binding.
  • S224P mutations in IgG4 Fc regions
  • M252Y/S254T/T256E mutations in IgGl may extend the half-life.
  • T366W in combination with T366S/L368A/Y407V mutations 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.
  • 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).
  • 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).
  • serum proteins that may act as stabilisation domain may include albumin, fibrinogen, fibronectin, haemoglobin, transferrin, an immunoglobulin domain.
  • 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.
  • 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.
  • 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:
  • the stabilisation domain comprises or consists of a polyethylene glycol (PEG) polymer.
  • the primary function of the stabilisation domain is to stabilise or to extend the half-life of the agent in a biological system.
  • the stabilisation domain may have a secondary function wherein the stabilisation domain has a clinical or therapeutically beneficial effect.
  • the agent according to the claimed invention is an antibody drug conjugate (ADC)
  • ADC antibody drug conjugate
  • 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.
  • 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 Biotechn Vietnamese Anlagens und Anlagensges M.B.H).
  • Fcabs antigen-binding domains in the CH3 domains
  • W02006/072620 and WO2009/132876 both F-Star Biotechn Vietnamese Anlagens und Anlagensges M.B.H.
  • 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.
  • the Fc domain comprises or consists of an Fcab.
  • 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.
  • 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.
  • proteolysis 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).
  • the cleavage site is not the amino acid sequence DEVD (SEQ ID NO: 151).
  • 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).
  • the cleavage site is located in a hinge region.
  • a hinge region we include the meaning of a hydrophilic sequence of the heavy chains of an antibody.
  • 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.
  • 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.
  • 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).
  • the cleavable domain is located in the upper hinge.
  • 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).
  • 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:
  • IgGl upper and middle hinge region (SEQ ID NO: 154): EPKSCDKTHTCPPCP
  • IgGl lower hinge region (SEQ ID NO: 155): (A)PELLGGPSVFLF
  • 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
  • Table D IgG4 lower hinge region (SEQ ID NO: 157): (A)PEFLGGPSVFLF
  • 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,
  • 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.
  • the C-terminal cysteine of the CHI domain is left intact.
  • “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.
  • the C-terminal cysteine of the CHI domain is not deleted or substituted.
  • 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.
  • the cleavable domain or cleavage site is located after the C-terminal cysteine of the CHI domain (i.e.
  • 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.
  • 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.
  • 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,).
  • the cleavable domain and/or the cleavage site may be located before the first C of the hinge motif CPPC (or CPSC).
  • 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.
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site ends at:
  • the cleavable domain and/or cleavage site is:
  • the cleavable domain and/or cleavage site is:
  • located we include that the cleavable domain and/or cleavage site is present at this location in the hinge.
  • 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.
  • position Z 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.
  • 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.
  • 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.
  • 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.
  • the cleavable domain and/or cleavage site is located after:
  • the cleavable domain and/or cleavage site is located before and/or ends at:
  • the cleavable domain and/or cleavage site ends at:
  • the cleavable domain and/or cleavage site is:
  • the cleavable domain and/or cleavage site is: • located after position 5 and ends at position 10, as defined by IMGT hinge numbering;
  • 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)):
  • the cleavable domain and/or cleavage site is:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site is:
  • cleavable domain e.g. the cleavage site and/or linker domain(s):
  • the cleavable domain and/or cleavage site may be:
  • cleavable domain e.g. the cleavage site and/or linker domain(s):
  • the cleavable domain or cleavage site is:
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site ends at:
  • the cleavable domain and/or cleavage site is:
  • the cleavable domain and/or cleavage site is:
  • the cleavable domain or cleavage site may be engineered into the hinge as an insertion between:
  • one, two, three, four or five of the amino acids in positions 5-10 are deleted or substituted by the cleavable domain (e.g. the cleavage site and/or linker domain(s)).
  • one, two, three, four or five of the amino acids in positions 5-10 are deleted.
  • one, two, three, four or five of the amino acids in positions 5-10 are substituted by the cleavable domain (e.g. the cleavage site and/or linker domain(s)).
  • position 1.6 IMGT unique numbering for C-domain CH2
  • 231 EU numbering
  • 244 Kabat numbering
  • 244 Chothia numbering
  • the agent comprises (native) amino acid residues at one or more (e.g. all) of the above positions.
  • 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.
  • the cleavable domain is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain ends at:
  • the cleavable domain is:
  • the cleavable domain is:
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site is:
  • the cleavable domain and/or cleavage site may be inserted in the hinge region between:
  • one, two or three of the residues in positions 5-7 are deleted.
  • positions 233-238 Kabat numbering
  • positions 230-238 Chothia numbering
  • one or more of the residues between positions 218- 226 exclusive are deleted, i.e. not including residue 218 or 226.
  • one, two, three, four or five of the amino acids in positions 5-10 are substituted.
  • the residues are substituted for residues in the cleavable domain, such residues in the cleavage site and/or linker domain.
  • the residues in one or more of the following positions are left intact:
  • IMGT hinge numbering IgG4 • position 12 (IMGT hinge numbering IgG4), 230 (EU numbering), 243 (Kabat numbering) and/or 243 (Chothia numbering).
  • the agent comprises (native) amino acid residues at one or more (e.g. all) of the above positions.
  • position 1.6 IMGT unique numbering for C-domain CH2
  • 231 EU numbering
  • 244 Kabat numbering
  • 244 Chothia numbering
  • the agent comprises (native) amino acid residues at one or more (e.g. all) of the above positions.
  • the cleavable domain and/or cleavage site is not located in the middle hinge.
  • 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).
  • middle hinge may be located from:
  • the middle hinge may be located from:
  • the middle hinge may comprise:
  • IMGT hinge numbering scheme • 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
  • IMGT hinge numbering scheme • 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
  • IMGT hinge numbering scheme • 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
  • IMGT hinge numbering scheme • 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).
  • 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).
  • position 11 IMGT hinge numbering scheme
  • position 226 EU numbering scheme
  • position 239 Kabat numbering scheme
  • position 239 Chothia numbering scheme
  • position 12 IMGT hinge numbering scheme
  • position 221 EU numbering scheme
  • position 240 Kabat numbering scheme
  • position 240 Chothia numbering scheme
  • position 13 IMGT hinge numbering scheme
  • position 228 EU numbering scheme
  • position 241 Kabat numbering scheme
  • position 241 Chothia numbering scheme
  • position 14 IMGT hinge numbering scheme
  • position 229 EU numbering scheme
  • position 242 Kabat numbering scheme
  • position 242 Chothia numbering scheme
  • the middle hinge may be located from
  • the middle hinge may comprise:
  • IMGT hinge numbering scheme • 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
  • IMGT hinge numbering scheme • 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
  • IMGT hinge numbering scheme • 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
  • IMGT hinge numbering scheme • 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).
  • the agent e.g. as part of the cleavable domain
  • position 8 IMGT hinge numbering scheme
  • position 226 EU numbering scheme
  • position 239 Kabat numbering scheme
  • position 239 Chothia numbering scheme
  • This position defines the start of the 'middle hinge' region necessary for the linkage of the two heavy chains.
  • position 9 IMGT hinge numbering scheme
  • position 221 EU numbering scheme
  • position 240 Kabat numbering scheme
  • position 240 Chothia numbering scheme
  • position 10 IMGT hinge numbering scheme
  • position 228 EU numbering scheme
  • position 241 Kabat numbering scheme
  • position 241 Chothia numbering scheme
  • position 11 IMGT hinge numbering scheme
  • position 229 EU numbering scheme
  • position 242 Kabat numbering scheme
  • position 242 Chothia numbering scheme
  • 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.
  • 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.
  • N-terminal proline of the lower hinge is left intact.
  • the agent comprises an N-terminal proline of the lower hinge.
  • 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.
  • 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).
  • 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.
  • 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.
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site ends at:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site is ends at:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site may be engineered into the hinge as an insertion between:
  • 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:
  • 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-
  • Kabat numbering and/or 256 Chothia numbering
  • position 4 IMGT unique numbering for C-domain CH2
  • EU numbering EU numbering
  • 257 Kabat numbering
  • Chothia numbering 257
  • the agent comprises (native) amino acid residues at one or more (e.g. all of) the above positions.
  • 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.
  • N-terminal proline of the lower hinge is left intact.
  • the agent comprises an N-terminal proline of the lower hinge.
  • 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.
  • 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).
  • 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.
  • 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.
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site ends at:
  • 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);
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site is located after:
  • cleavable domain and/or cleavage site is located before:
  • the cleavable domain and/or cleavage site is ends at:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site may be:
  • the cleavable domain and/or cleavage site may be engineered into the hinge as an insertion between:
  • 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:
  • 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:
  • the protease that acts on the cleavage site is expressed by and/or accumulates in the vicinity of unwanted cells or a tumour.
  • the protease is a tumourspecific protease.
  • the cleavage site may be cleavable selectively by proteases that are found in the vicinity of the tumour cells.
  • Specificity of the agent may be increased by virtue of the cleavage site only being cleaved in the vicinity of the unwanted cells.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • cysteine protease including the Cathepsin family B, L, S etc
  • an aspartyl or aspartic protease including Cathepsin D and E, or Napsin A
  • a serine protease including Cathepsin A and G, Thrombin, Plasmin, uPA, tissue Plasminogen Activator (tPA), matriptase, MT-SP1, fibroblast activation protein (FAP)
  • 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).
  • MMP1-28 metalloproteinase
  • the protease may belong to the A Disintegrin and Metalloproteinase (ADAM) and A Disintegrin, or Metalloproteinase with Thrombospondin Motifs (ADAMTS) families of proteases.
  • ADAM A Disintegrin and Metalloproteinase
  • ADAMTS Metalloproteinase with Thrombospondin Motifs
  • 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
  • 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.
  • 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).
  • the cleavable domain may comprise a uPA protease cleavage site comprising or consisting of the sequence: LSGRSDNH (SEQ ID NO: 77).
  • 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.
  • 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)).
  • the cleavable domain may comprise an ADAM10 protease cleavage site comprising or consisting of the sequence: PRAEALKGG (SEQ ID NO: 89).
  • 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.
  • the cleavable domain comprises one or more linker domains present between the therapeutic domain and the stabilisation domain.
  • 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).
  • 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(G 4 S) 4
  • 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.
  • 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.
  • 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.
  • the linker domain comprises or consists of neutral amino acids.
  • at least 70%, 80%, 90%, 95% or 100% of the amino acids in the linker domain are neutral amino acids.
  • neutral amino acids we include amino acids that possess an equal number of amino and carboxylic acid groups.
  • neutral amino acids include glycine, alanine, leucine, isoleucine, valine, phenylalanine, proline, methionine, serine, threonine, tyrosine, glutamine, asparagine and tryptophan.
  • the linker domain comprises or consists of S, G, A and/or T.
  • at least 70%, 80%, 90%, 95% or 100% of the amino acids in the linker domain are S, G, A or T.
  • 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.
  • the linker domain comprises spacer sequences comprising A, G and/or T.
  • 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.
  • the agent comprises a His-tag (HHHHHH, SEQ ID NO: 143).
  • 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.
  • the cleavable domain may be an additional sequence (which adds further amino acid to the biologic).
  • 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).
  • it could be a combination of the addition of a sequence and mutagenesis.
  • 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.
  • small cleavage sites or small cleavable domains are favourable because the risk of immunogenicity (to foreign/non-host sequences) may be lower.
  • the at least one cleavage site and/or the cleavable domain is 4 to 20 amino acids in length.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • MMP-2 Gelatinase A
  • MMP-3 Stromelysin 1
  • MMP-7 Matrilysin
  • MMP-9 Gelatinase B
  • MMP-12 Macrophage metalloelastase
  • MMP-13 Collagenase-3
  • Cathepsin G9 Cathepsin G9
  • the cleavable domain is not cleavable by the protease Capsase-3 or by the PreScission protease.
  • PreScission protease we include the meaning of
  • the therapeutic domain is a Fab
  • the cleavable domain comprises a uPA cleavage site
  • the stabilisation domain is an Fc region
  • the therapeutic domain is a Fab
  • the cleavable domain comprises a ADAM10 cleavage site
  • the stabilisation domain is an Fc region
  • the therapeutic domain is a VHH
  • the cleavable domain comprises a uPA cleavage site
  • the stabilisation domain is an Fc region
  • the therapeutic domain is a ScFv
  • the cleavable domain comprises a uPA cleavage site
  • the stabilisation domain is an Fc region.
  • the therapeutic domain is a scFv
  • the cleavable domain comprises a uPA cleavage site
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the stabilisation domain does not comprise or consist of a sequence of SEQ ID NO: 27, 28, 29 and/or 101.
  • 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.
  • the agent does not comprise:
  • 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;
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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,
  • 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,
  • the agent does not comprise a cleavable domain and/or cleavage site comprising a sequence of SEQ ID NO: 77 or 89.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the agent does not comprise or consist of a sequence of any one of SEQ ID NOs: 1-81, 89, 90 and/or 92.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the combinations of SEQ ID NOs of the heavy chain and light chain are as per the agents that are described in the examples.
  • ADCs antibody- drug-conjugates
  • ADCs 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).
  • 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.
  • ADC antibody-drug-conjugate
  • 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.
  • the payload delivers a therapeutic effect directly to the cancer cells while minimizing damage to healthy tissues.
  • a therapeutic domain comprises the payload.
  • the payload is linked to the therapeutic domain.
  • Paymentload may further comprise detectable agents, for example a dye or a radioactive tracer.
  • the "payload” may be replaced with a detectable agent, for example a dye or a radioactive tracer.
  • 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.
  • chemotherapeutic agent we include a drug (medicament or pharmaceutically active ingredient) that has efficacy in the treatment of cancer.
  • the one or more payloads may be conjugated to the therapeutic domain, the cleavable domain and/or the stabilisation domain.
  • 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.
  • 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.
  • the one or more payloads is attached to the agent via native or engineered cysteine conjugation.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • ADC linkers examples include cleavable linkers, and include (but is not limited to):
  • valine-citrulline (cleavable and protease sensitive);
  • N-maleimidomethylcyclohexane-l-carboxylate (non-cleavable);
  • the invention relates to a method for preparing an antibody-drug-conjugate comprising:
  • the one or more payload may be conjugated to the therapeutic domain, cleavable domain and/or stabilisation domain of the agent.
  • 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.
  • 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.
  • conjugation ADC linkers and payloads in the methods may be as described elsewhere herein, for example as for the previous aspects of the invention.
  • the invention relates to an ADC produced by the method of the third aspect.
  • the invention provides a pharmaceutical composition, comprising an agent according to the invention, and optionally a pharmaceutically acceptable carrier, diluent or excipient.
  • the invention provides a pharmaceutical composition comprising an ADC according to the invention, and optionally a pharmaceutically acceptable carrier, diluent or excipient.
  • 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.
  • 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.
  • 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.
  • a pharmaceutically acceptable composition is manufactured according to GMP standards.
  • 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.
  • buffer we include a solution that can resist pH change upon the addition of an acidic or basic components.
  • the buffer may be (but is not limited to) a citrate buffer, a phosphate buffer and/or an acetate buffer.
  • isotonic agent we include a solution that has the same concentration of solutes as another solution (e.g. as the blood).
  • the isotonic agent may be (but is not limited to) sodium chloride (e.g. saline solution or Ringer solution).
  • 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.
  • the antioxidant may be (but is not limited to) ascorbic acid or cysteine.
  • chelator we include a compound that can bind metal ions, such as ethylenediaminetetraacetic Acid (EDTA).
  • 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.
  • citric acid dextrose, acetic acid
  • glutamic acid glycine
  • L-histidine L-histidine monohydrochloride monohydrate
  • 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.
  • 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.
  • 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).
  • the bag is an intravenous (IV) bag.
  • the bag, syringe or injection device may be for intravenous, subcutaneous, intramuscular, intradermal and/or intraosseous delivery.
  • the bag, syringe or injection device is for intravenous or subcutaneous delivery.
  • the invention provides a kit 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 (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
  • ADC antibody-drug conjugate
  • 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).
  • a marketing authorisation number e.g., an FDA or EMA authorisation number, or an authorisation number for a country of interest.
  • 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.
  • 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.
  • the cancer is a solid tumour.
  • the cancer may be as defined elsewhere in the application.
  • the kit further comprises an IV bag, syringe or injection device that comprises the agent.
  • 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.
  • 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.
  • the kit further comprises packaging.
  • 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.
  • 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.
  • therapy such as in the treatment or prevention of a tumour, optionally of a cancer.
  • 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.
  • treatment denote an approach for obtaining a beneficial or desired result including and preferably a beneficial or desired clinical result.
  • 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.
  • the agent 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).
  • 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.
  • 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.
  • the vicinity of unwanted cells or the tumour comprises a high titre and/or a high activity of tumour-specific proteases.
  • 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.
  • an appropriate assay to measure titre and activity of the protease for example ELISA, activitybased protein profiling (ABPP), mass spectrometry, fluorescent probe imaging.
  • protease activity is measured by an ELISA assay.
  • the subject of treatment or prevention is a mammal, preferably a human.
  • 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.
  • the tumour is a solid tumour.
  • 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
  • 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).
  • 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).
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • a cell culture system such as mammalian cells, bacteria, yeast or insect cells.
  • 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.
  • 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.
  • 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.
  • the agents, ADCs or pharmaceutical compositions of the invention and the additional agent(s) are formulated in a single composition.
  • the agent, ADC or pharmaceutical composition of the invention and the additional agent(s) are administered as two or more separate compositions.
  • the agent, ADC or pharmaceutical composition of the invention and the additional agent(s) are administered simultaneously.
  • the agent, ADC or pharmaceutical composition of the invention and the additional agent(s) are administered sequentially.
  • a particular protease inhibitor so as to improve the target selectivity of the agent or ADC of the invention.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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).
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • antibodies such as anti-CHl antibodies, or anti-CLl antibodies
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • labelling such as labelling the Fab region of an antibody with a fluorophore
  • detection of the fluorophore using an antibody such as detection of the fluorophore using an antibody
  • immunostaining such as anti-CHl antibodies, anti-lambda antibodies, or anti-kappa antibodies
  • 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.
  • 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.
  • the parts of the agent 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.
  • 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.
  • 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.
  • the parts of the ADC 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.
  • 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.
  • 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:
  • a stabilisation domain optionally wherein the agent is an ADC which further comprises one or more payloads and optionally one or more ADC linkers.
  • step (b) comprises determining (or having determined) whether the sample comprises the antigen of interest and the protease of interest.
  • 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.
  • ELISAs enzyme-linked immunosorbent assays
  • LFIAs lateral flow immunoassays
  • 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).
  • 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.
  • 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.
  • 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.
  • 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.
  • the invention provides a method for introducing a therapeutic domain into a tumour microenvironment in a subject, wherein the method comprises:
  • the reduction of size in the vicinity of the tumour microenvironment may improve the penetrability of the therapeutic domain into the tumour.
  • the invention provides a method for reducing the size of an agent or ADC in vivo, comprising:
  • agent or ADC as described elsewhere herein, wherein the agent or ADC comprises a cleavage site
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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:
  • 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).
  • 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).
  • 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)).
  • CPPC hinge disulphide bond region
  • 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.
  • 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).

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Abstract

La présente invention concerne des procédés et des conjugués pour améliorer la pénétrabilité de substances biologiques dans le micro-environnement tumoral (TME) à des fins thérapeutiques, l'agent comprenant de manière appropriée au moins : i. un domaine thérapeutique ; ii. un domaine clivable ; et iii. un domaine de stabilisation.
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