WO2025062139A1

WO2025062139A1 - A conjugate of the c-terminal domain of pcsk9 and an anti-receptor antibody or antigen-binding fragment thereof

Info

Publication number: WO2025062139A1
Application number: PCT/GB2024/052428
Authority: WO
Inventors: Bernard Kelly; David Owen; Stephen Graham; John Sinclair
Original assignee: Cambridge Enterprise Ltd
Current assignee: Cambridge Enterprise Ltd
Priority date: 2023-09-20
Filing date: 2024-09-20
Publication date: 2025-03-27
Anticipated expiration: 2026-03-20
Also published as: GB202314424D0

Abstract

The present invention relates to a conjugate, in particular to a conjugate comprising an antigen- binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof, wherein the antigen-binding moiety is operable to bind to a receptor on the surface of a cell, and wherein the receptor is constitutively internalised and recycled. The conjugates may be used in methods for degrading a cell surface receptor and/or inhibiting the growth of a cell.

Description

A CONJUGATE OF THE C-TERMINAL DOMAIN OF PCSK9 AND AN ANTI-RECEPTOR ANTIBODY OR ANTIGEN-BINDING FRAGMENT THEREOF

FIELD

[01] The present invention relates to a conjugate, in particular to a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof, wherein the antigen-binding moiety is operable to bind to a receptor on the surface of a cell, and wherein the receptor is constitutively internalised and recycled. The conjugates may be used in methods for degrading a cell surface receptor and/or inhibiting the growth of a cell.

BACKGROUND

[02] Antibodies are well-known for their target specificity and are used as therapeutics to treat a wide variety of human diseases. Because of their large size (~150kDa) and inability to cross the plasma membrane (PM), antibodies are generally not able to engage cytoplasmic targets in vivo, being instead well-suited to targeting secreted proteins. Typically, antibody therapeutics work by blockade of a functional binding surface, by recruitment of immune effector functions leading to target cell killing (ADCC, antibody-dependent cellular cytotoxicity), or by mediating endolysosomal delivery of cytotoxic drug payloads (ADCs, antibody-drug conjugates) (Drago et al, Nat Rev Clin Oncol 18: 327-344, 2021). Nonetheless, many potential transmembrane protein targets have proven difficult to drug effectively with antibodies, and some targets of ADCs evade delivery to endolysosomes by efficient recycling to the PM (Hammood et al, Pharmaceuticals 14: 674, 2021).

[03] Recent advances have used antibodies to drive target degradation, inspired by the concept of Proteolysis Targeting Chimeras (PROTACs). PROTACs, which are heterobifunctional small molecule compounds consisting of a ligand for the target protein, a linker, and a ligand to recruit E3 ligase, have been utilised to induce degradation of the target proteins. PROTACs forcibly recruit cytoplasmic E3 ubiquitin ligases to a target to stimulate its ubiquitination and subsequent proteasomal degradation. It has thus emerged as an alternative approach to target 'undruggable' cytoplasmic proteins.

[04] Polyubiquitination of the cytosolic domains of transmembrane proteins marks them for degradation in endolysosomes rather than proteasomes, a process mediated by ESCRTs (Endosomal Sorting Complexes Required for Transport). AbTACS/PROTABs (Marei et al, 2022; Cotton et al, 2021) take advantage of this mechanism by forcibly recruiting transmembrane E3 ubiquitin ligases to target receptors with bispecific antibodies, resulting in ubiquitination of the target's cytosolic domains and subsequent endolysosomal degradation. An alternative approach utilises LYTACs (lysosome-targeting chimaeras) to hijack an endogenous mechanism for delivery of lysosomal hydrolases by labelling antibodies with sugars that are recognized by the cationindependent mannose-6-phosphate receptor (CIMPR). This leads to co-trafficking of the antibody and its bound target, together with CIMPR, to endolysosomes, where the target is degraded. [05] However, there remains a need to expand the antibody-mediated protein degradation toolbox to provide more effective therapies for treating a wide range of diseases.

[06] The subcellular localisation of transmembrane (TM) proteins is tightly regulated by the endomembrane trafficking system. Many TM proteins traffic through, or are localised to, the plasma membrane (PM). Of these TM proteins, a subset undergo a continual process of internalisation from the PM followed by recycling back to the PM. Their steady-state localisation can be endosomal or PM depending on the kinetics of their recycling.

[07] Internalisation from the PM is most commonly effected by clathrin-mediated endocytosis. In this process, TM proteins are packaged into clathrin-coated vesicles that bud from the PM and migrate to the cell interior for onward processing. Often, TM proteins are marked as 'cargo' for clathrin-mediated endocytosis by the presence of short, linear amino acid motifs present in the unstructured parts of their cytosolic regions. These motifs are recognised and bound by 'clathrin adaptors', which simultaneously bind clathrin in order to draw the cargo into clathrin-coated vesicles as they form.

[08] Once TM proteins have been internalised into clathrin-coated vesicles, the vesicles fuse to form early endosomes. Early endosomes undergo a complex process of maturation to become 'sorting endosomes'. A subset of TM proteins that have been internalised are trafficked back to the Golgi I trans-Golgi via the clathrin adaptor complex AP1 . Other TM proteins are recycled to the PM, and this is an important but relatively poorly-understood process. In it, TM proteins are selected as cargo to be packaged into 'tubules' that bud off from sorting endosomes and are transported back to the PM, where they fuse. Several different protein complexes are known to be involved in this process, including the retromer and retriever complexes. It is likely that these complexes recognise motifs in the cytosolic portions of the TM proteins.

SUMMARY

[09] According to a first aspect of the present invention there is provided a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof; wherein the antigen-binding moiety is operable to bind to a receptor on the surface of a cell; and wherein the receptor is constitutively internalised and recycled.

[10] According to a second aspect of the present invention there is provided a method of degrading a cell surface receptor that is constitutively internalised and recycled, the method comprising contacting the receptor with a conjugate according to the first aspect of the present invention; wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.

[11 ] According to a third aspect of the present invention there is provided a method of inhibiting the growth of a cell having a receptor on the surface thereof, the method comprising contacting the cell with a conjugate according to the first aspect of the present invention; wherein the receptor is constitutively internalised and recycled; wherein the antigen-binding moiety is operable to bind to the receptor; and wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.

[12] According to a fourth aspect of the present invention there is provided the use of a conjugate according to the first aspect of the present invention to degrade a cell surface receptor that is constitutively internalised and recycled.

[13] According to a fifth aspect of the present invention there is provided the use of a conjugate according to the first aspect of the present invention to inhibit the growth of a cell having receptor on the surface thereof; wherein the receptor is constitutively internalised and recycled; and wherein the antigen-binding moiety is operable to bind to the receptor.

[14] According to a sixth aspect of the present invention there is provided a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9), wherein the C-terminal domain of PCSK9 consists of an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO. 39.

BRIEF DESCRIPTION OF DRAWINGS

[15] Figure 1 shows (a) a GFP immunoblot detecting the presence of TfR-GFP in HeLa cells treated with PCTD-aGFP and aGFP alone; (b) shows TfR-GFP degradation assessed by densitometry. Data shown as Superplot: biological replicates differ by shape; large symbols indicate means. Differences in means were assessed by one-way ANOVA with Tukey's method for multiple comparisons: ns, p > 0.05; *, p < 0.05; **, p < 0.01 ; (c) shows an immunoblot detecting the presence of endogenous TfR in HeLa cells treated with OKT9FabPCTD and OKT9FabHA; and (d) shows TfR degradation assessed by densitometry (quantitation as in (c)).

[16] Figure 2 shows a schematic of reporter constructs comprising an extracellular CD8 a-chain tagged with the BC2 peptide, a single transmembrane helix, a variable cytoplasmic domain, and an intracellular EGFP for fluorescence detection.

[17] Figure 3 shows flow cytometric analysis for different reporter HeLa cell lines treated with anti-CD8 antibody or left untreated. Traces are depicted for a representative sample from two biological replicate experiments; red traces depict cells exposed to anti-CD8 antibody and blue traces depict untreated cells.

[18] Figure 4 shows (a) flow cytometric analysis of PCTD-mediated degradation. Representative traces from at least three biological replicates; and (b) Log2-fold change in mean fluorescence with treatment compared to mean fluorescence of untreated is depicted for all experiments as a Superplot. Differences in means were assessed by one-way ANOVA with Tukey's method for multiple comparisons: ns, p > 0.05; *, p < 0.05; **, p < 0.01 . [19] Figure 5 shows the dependence of PCTD-mediated degradation on lysosomal acidification. NPXY reporter cells were pre-incubated with DMSO (a) or Bafilomycin A1 (b) for 2 hours, treated overnight with PCTD-aBC2 (red) or PBS (grey), and cellular GFP fluorescence analysed by flow cytometry. Data representative of two biological replicates.

[20] Figure 6 shows a quantified normalised spot intensity of fibroblasts infected with US28- GFP and, three days post-infection, treated with PCTD-Vun100bv, with a non-US28-targeting PCTD-aBC2 control, or left untreated, and imaged using live-cell fluorescence microscopy. The data are representative of at least 5 technical replicates per condition.

[21 ] Figure 7 shows flow cytometric analysis for fibroblasts infected with US28-GFP-HCMV and which were left untreated or were treated with Vunl OObv or PCTD-Vun100bv three days postinfection.

[22] Figure 8 shows relative fluorescence intensity data for Kasumi-3 cells infected with US28- GFP treated with PCTD-Vun100bv or left untreated five days post-infection.

[23] Figure 9 shows IE-positive nuclei counts data for kasumi-3 cells infected with HCMV-IE2- eYFP and, two hours post-infection, treated with VunlOObv, PCTD-Vun100bv, or PMA (a) three and (b) four days post-treatment. Representative figures from four (a) or three (b) biological replicates are shown.

[24] Figure 10 shows flow cytometric analysis of a HeLa cell line stably expressing a NPXY reporter construct treated overnight with PCTD-aBC2, PCTD(H553R and Q554R)-aBC2 or left untreated.

[25] Figure 11 shows Western blot analysis results (Licor fluorescence detection using antibody 29D8 and goat anti-rabbit Alexa Fluor 680, normalised with anti-GAPDH antibody and goat antimouse Alexa Fluor 780) of SKBR3 cells treated with trastuzumab, a trastuzumab-PCTD fusion, or left untreated. Bands were quantitated by densitometry using Fiji. Means and standard deviation from 2 experiments shown.

[26] Figure 12 shows flow cytometric analysis of a HeLa cell line stably expressing a NPXY reporter construct treated overnight with M1-aBC2, PCTD-aBC2 or left untreated.

[27] Figure 13 shows flow cytometry analysis of a HeLa cell line stably expressing a NPXY reporter construct treated overnight with M1SOL1-aBC2 or PCTD-aBC2 or left untreated.

[28] Figure 14 shows flow cytometry analysis of a HeLa cell line stably expressing a NPXY reporter construct treated overnight with M1-M2-aBC2, M1-M3-aBC2 or PCTD-aBC2 or left untreated.

[29] Figure 15 shows Western blot analysis results (chemiluminescence detection using antiactin goat primary AbCam, ab8229, anti-GFP rabbit primary AbCam, ab6556, and HRP anti-goat or anti-rabbit secondary antibody) of CD34+ cells treated with VunlOObv, PCTD-Vun100bv, or left untreated. Bands were quantitated by densitometry and plotted relative to the actin control and untreated cells. Mean values from triplicate samples shown.

DESCRIPTION OF EMBODIMENTS [30] The present inventors have found that the specificity of antibodies can be harnessed to effect targeted protein degradation of membrane proteins. It has surprisingly been demonstrated that targeted protein removal can be effected utilising the C-terminal domain of Proprotein Convertase Subtilisin/Kexin type 9 (PCSK9). PCSK9 suppresses low-density lipoprotein (LDL) receptor (LDLR, responsible for LDL-cholesterol homeostasis) by a poorly understood mechanism that reroutes LDLR to lysosomes for degradation. The N-terminal domain of PCSK9 binds tightly to LDLR at the plasma membrane (PM) and PCSK9 lacking the Cys/His-rich C- terminal domain is unable to stimulate LDLR degradation (Zhang, D.-W., Garuti, R., Tang, W.-J., Cohen, J. C. & Hobbs, H. H. Structural requirements for PCSK9-mediated degradation of the low- density lipoprotein receptor. Proc. Natl. Acad. Sci. U. S. A. 105, 13045-13050 (2008)). Recombinant antibodies genetically fused to the C-terminal domain of PCSK9 have surprisingly been found to drive degradation of receptor proteins that undergo constitutive internalisation and recycling, including the transferrin receptor and the human cytomegalovirus protein US28, for example.

[31] The present invention will now be further described. In the following passages, different aspects of the invention are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature indicated as being preferred or advantageous may be combined with any other feature or features indicated as being preferred or advantageous.

[32] Generally, nomenclatures used in connection with, and techniques of, cell and tissue culture, pathology, oncology, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art. The methods and techniques of the present disclosure are generally performed according to conventional methods well-known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated. See, e.g., Green and Sambrook et al., Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); Therapeutic Monoclonal Antibodies: From Bench to Clinic, Zhiqiang An (Editor), Wiley, (2009); and Antibody Engineering, 2nd Ed., Vols 1 and 2, Ontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[33] Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly accomplished in the art or as described herein. The nomenclatures used in connection with, and the laboratory procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well-known and commonly used in the art. Standard techniques are used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients. Suitable assays to measure the properties of the molecules disclosed herein are also described in the examples. Conjugate

[34] The conjugate of the present invention comprises an antigen-binding moiety. Suitably, the antigen-binding moiety is capable of binding to an antigen. The antigen-binding moiety may be operable to bind to a receptor on the surface of a cell. The receptor may be constitutively internalised and recycled.

[35] Suitably, the antigen-binding moiety may be capable of binding to a suitable receptor on the surface of a cell.

[36] The terms “antigen(s)” and “epitope(s)” are well established in the art and refer to the portion of a protein or polypeptide which is specifically recognized by a component of the immune system, e.g., an antibody or a T-cell I B-cell antigen receptor. As used herein, the term “antigen(s)” encompasses antigenic epitopes, e.g., fragments of antigens which are recognized by, and bind to, immune components. Epitopes can be recognized by antibodies in solution, e.g., free from other molecules. Epitopes can also be recognized by T-cell antigen receptors when the epitope is associated with a class I or class II major histocompatibility complex molecule.

[37] The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen to which an immunoglobulin, antibody or antigen-binding fragment thereof specifically binds. Generally, an antigen has several or many different epitopes and reacts with many different antibodies. The term “specifically” includes linear epitopes and conformational epitopes.

[38] Epitopes within protein antigens can be formed both from contiguous amino acids (usually a linear epitope) or non-contiguous amino acids juxtaposed by tertiary folding of the protein (usually a conformational epitope). Epitopes formed from contiguous amino acids are typically, but not always, retained on exposure to denaturing solvents, whereas epitopes formed by tertiary folding are typically lost on treatment with denaturing solvents. An epitope typically includes at least 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14 or 15 amino acids in a unique spatial conformation. Methods for determining what epitopes are bound by a given antibody or antigen-binding fragment thereof (i.e., epitope mapping) are well known in the art and include, for example, immunoblotting and immunoprecipitation assays, wherein overlapping or contiguous peptides from are tested for reactivity with a given antibody or antigen-binding fragment thereof. Competition assays can also be used to determine if a test antibody binds to the same epitope as a reference antibody. Suitable competition assays are mentioned elsewhere herein and also shown in the examples. In some aspects, the epitope to which an antibody or antigen-binding fragment thereof binds can be determined by, e.g., NMR spectroscopy, X-ray diffraction crystallography studies, ELISA assays, hydrogen/deuterium exchange coupled with mass spectrometry (e.g., liquid chromatography electrospray mass spectrometry), array-based oligo-peptide scanning assays, mutagenesis mapping (e.g, site-directed mutagenesis mapping), and/or in silico modelling.

[39] The term "antibody" as used herein refers to an immunoglobulin (Ig) protein that is capable of binding an antigen. In particular, the term "antibody" as used herein broadly refers to any polypeptide comprising complementarity determining regions (CDRs) that confer specific binding affinity of the polypeptide for an antigen. The term antibody as used herein encompasses polyclonal and monoclonal antibody preparations.

[40] As antibodies can be modified in a number of ways, the term "antibody" should be construed as covering antibody fragments, derivatives, functional equivalents and homologs of antibodies, including any polypeptide comprising an immunoglobulin binding domain. The term “antibody” should also be construed as covering antibody mimetics, such as, but not limited to, cyclic peptides, for example bicyclic peptides, cysteine knots and anticalins etc. The antibody may be an immunoglobulin (Ig) molecule, or antigen binding portion/fragment thereof, comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such fragments are known in the art for example F(ab')2, Fab, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. Therefore, an antibody fragment comprises an antigen binding portion. The present invention extends to such antibody fragments.

[41] The term "monoclonal antibody" refers to an antibody obtained from a single close of cells or cell line. The individual antibodies are identical and/or bind the same epitope. Unlike polyclonal antibodies, which include different antibodies directed against different epitopes, each monoclonal antibody in a preparation is directed against a single epitope.

[42] The antibody, or antigen-binding fragment thereof, described herein, "which binds" or is “capable of binding” an antigen, binds the antigen with sufficient affinity such that the antibody or antigen-binding fragment thereof may be useful as a therapeutic or diagnostic agent. The term "specific" may refer to the situation in which the antibody or antigen-binding fragment will not show any significant binding to molecules other than its specific binding partner.

[43] “Specifically binds", "specific binding" or "selective binding" means that the binding is selective for the antigen and can be discriminated from unwanted or non-specific interactions. The ability of an antigen binding moiety to bind to a specific antigenic determinant can be measured either through an enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed on a BIAcore instrument), and traditional binding assays. The binding reaction may be shown with reference to a negative control test using an antibody of unrelated specificity.

[44] The terms “polypeptide(s)” and “protein(s)” are used interchangeably throughout the application and denote at least two covalently attached amino acids, thus may signify proteins, polypeptides, oligopeptides, peptides, and fragments thereof. The protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Hence, “amino acid(s)” or “peptide residue(s)”, as used herein, denote both naturally occurring and synthetic amino acids. In some cases, the immunoglobulin proteins of the present invention may be synthesized using any in vivo or in vitro protein synthesis technique known in the art. [45] A full-length antibody comprises two heavy (H) chains and two light (L) chains. Each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR, VH or VH) and a heavy chain constant region. The heavy chain constant region is comprised of three domains, CH1 , CH2 and CH3. Each light chain is comprised of a light chain variable region or domain (abbreviated herein as LCVR, VL or VL) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[46] The heavy chain and light chain variable regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each heavy chain and light chain variable region is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1 , CDR1 , FR2, CDR2, FR3, CDR3, FR4.

[47] Immunoglobulin molecules can be subdivided into various types class and subclass. In humans the classes (isotype) of immunoglobulin include IgG, IgM, IgA, IgE, and IgD. The immunoglobulin classes are distinguished by the type of heavy chain they contain. IgG molecules possess heavy chains known as y-chains; IgM have p-chains; IgA have a-chains; IgE have e- chains; and IgD have b-chains.

[48] Antibodies may include the kappa (K) and lambda (A) light chains and the alpha (IgA), gamma (lgG1 , lgG2, lgG3, lgG4), delta (IgD), epsilon (IgE) and mu (IgM) heavy chains, or their equivalents in other species. Full-length immunoglobulin “light chains” (usually of about 25 kDa or 214 amino acids long) consist of a variable region of approximately 110 amino acids at the NH2-terminus and a kappa or lambda constant region at the COOH-terminus. Full-length immunoglobulin “heavy chains” (usually of about 50 kDa or 446 amino acids long), likewise consist of a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).

[49] Light or heavy chain variable regions are generally composed of a “framework” region (FR) interrupted by three hypervariable regions, also called CDRs. The extent of the framework region and CDRs have been precisely defined. The sequences of the framework regions of different light and heavy chains are relatively conserved within a species. The framework region of an antibody, i.e., the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs. The CDRs are primarily responsible for binding to an epitope of an antigen. There are three CDRs in each of the variable regions of the heavy chain and the light chain, which are designated CDR1 , CDR2 and CDR3, for each of the variable regions. The term "CDR set" refers to a group of three CDRs that occur in a single variable region capable of binding the antigen. The exact boundaries of these CDRs can be defined differently according to different systems known in the art.

[50] Heavy chain CDRs are designated HCDR1 , HCDR2 and HCDR3. Light chain CDRs are designated LCDR1 , LCDR2 and LCDR3.

[51] The antibody may be comprised of four polypeptide chains, two heavy (H) chains and two light (L) chains, or any functional fragment, mutant, variant, or derivation thereof, which retains the essential epitope binding features of an Ig molecule. Such mutant, variant, or derivative antibody formats are known in the art.

[52] The term "CDR" (complementarity-determining region) refers to the immunoglobulin hypervariable domains within an antibody or antigen binding fragment sequences. It is generally accepted that the CDRs determine the specific antibody binding.

[53] Different definitions of the CDRs are commonly in use. The method described by Kabat is the most commonly used and CDRs are based on sequence variability (Kabat et al., (1971) Ann. NY Acad. Sci. 190:382-391 and Kabat, et al., (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91- 3242). Chothia refers instead to the location of the structural loops (Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987)). The Kabat numbering system is generally used when referring to a residue in the variable domain (approximately residues 1-107 of the light chain and residues 1 - 113 of the heavy chain). Another system is the ImMunoGeneTics (IMGT) numbering scheme (Lefranc et al., Dev. Comp. Immunol., 29, 185-203 (2005)). According to the IMGT numbering scheme, a CDR is a loop region of a variable domain, delimited according to the IMGT unique numbering for V domain. There are three CDR-IMGT in a variable domain: CDR1-IMGT (loop BC), CDR2-IMGT (loop C'C"), and CDR3-IMGT (loop FG).

[54] The Kabat system as described above is used herein, unless otherwise stated. The terms "Kabat numbering", " Kabat definitions" and " Kabat labelling" are used interchangeably herein.

[55] The term "antibody" is not only inclusive of antibodies generated by methods comprising immunisation, but also includes any polypeptide, e.g., a recombinantly expressed polypeptide, which is made to encompass at least one CDR capable of specifically binding to an epitope on an antigen of interest. Hence, the term applies to such molecules regardless whether they are produced in vitro, in cell culture, or in vivo. Methods of producing polyclonal and monoclonal antibodies are known in the art and described more fully below.

[56] It is possible to take monoclonal and other antibodies and use techniques of recombinant DNA technology to produce other antibodies or chimeric molecules which generally retain the specificity of the original antibody. Such techniques may involve introducing the CDRs into a different immunoglobulin framework, or grafting variable regions onto a different immunoglobulin constant regions. Alternatively, a hybridoma or other cell producing an antibody molecule may be subject to genetic mutation or other changes, which may or may not alter the binding specificity of antibodies produced.

[57] The antibody or antigen-binding fragment thereof may be chimeric, human or humanised. A “chimeric antibody” is a recombinant protein that contains the variable domains including the CDRs of an antibody derived from one species, for example a murine antibody, while the constant domains of the antibody molecule are derived from those of a different species, for example a human antibody. Methods to humanise antibodies include CDR grafting based on framework regions homology and antibody resurfacing. Human or humanised antibodies or antigen-binding fragments are most desirable for use in antibody diagnostics or therapies, as such molecules would elicit little or no immune response in the human subject.

[58] A humanised antibody is a recombinant protein in which the CDRs from an antibody from one species; e.g., a rodent antibody, are transferred from the heavy and light variable chains of the rodent antibody into human heavy and light variable domains (e.g., framework region sequences). The constant domains of the antibody molecule are derived from those of a human antibody.

[59] The term invention includes antibodies and antigen-binding fragments thereof. The antigen-binding fragments may be selected from any fragment capable of binding the antigen or antigenic fragment of interest. Exemplary antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, F(ab')3, Fabc, Fd, single chain Fv (scFv), (scFv)2, Fv, scFv-Fc, heavy chain only antibody, diabody, tetrabody, triabody, minibody, antibody mimetic protein, single domain antibody, e.g. a VH. Thus, the antigen-binding fragment may comprise or consist of any of these fragments.

[60] Antigen-binding fragments derived from an antibody, including single-chain antibodies, may comprise the variable region(s) alone or in combination with the entire, or parts of the, following: a heavy chain constant domain, or a portion thereof, e.g. a CH1 , CH2, CH3, transmembrane, and/or cytoplasmic domain, on the heavy chain, and a light chain constant domain, e.g. a Ckappa or Clambda domain, or portion thereof on the light chain. Also included in the present disclosure are any combinations of variable region(s) and CH1 , CH2, CH3, Ckappa, Clambda, transmembrane and cytoplasmic domains.

[61] Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv).

[62] The smallest antigen-binding fragment is the single variable fragment, namely the variable heavy (VH) or variable light (VL) chain domain. VH and VL domains respectively are capable of binding to an antigen. They are generally referred to as a “single domain antibody” or “immunoglobulin single variable domain”. A single domain antibody (~12 to 15 kDa) has thus either the VH or VL domain. Antigen-binding single VH domains have also been identified from, for example, a library of murine VH genes amplified from genomic DNA from the spleens of immunized mice and expressed in E. coli (Ward et al., 1989, Nature 341 : 544-546). Ward et al. named the isolated single VH domains "dAbs," for "domain antibodies." The term "dAb" or “sdAb” (for single domain antibody) generally refers to a single immunoglobulin variable domain (VH, VHH or VL) polypeptide that specifically binds antigen. For use in therapy, human single domain antibodies are preferred over camelid derived VHH, primarily because they are not as likely to provoke an immune response when administered to a patient.

[63] A "Fab molecule" (fragment antigen binding) as such a Fab domain refers to a protein consisting of the VH and CHI domain of the heavy chain (the "Fab heavy chain") and the VL and CL domain of the light chain (the "Fab light chain") of an immunoglobulin. In certain embodiments the Fab light chain and Fab heavy chain in the Fab construct are linked by a polypeptide sequence to yield a single chain Fab (scFab).

[64] A “Fab” or “Fab fragment” comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains. For example a Fab contains the constant domain (CL) of the light chain and the first constant domain (CHI) of the heavy chain along with the variable domains VL and VH on the light and heavy chains respectively. The variable domains comprise the complementarity determining loops (CDR, also referred to as hypervariable region) that are involved in antigen binding. A Fab’ comprises an antigen-binding domain comprising or consisting of one constant and one variable domain of each of the heavy and the light chains and the thiol group which forms the disulphide bridge between two heavy chains in a full-length antibody. The antigen binding fragment may comprise a Fab. The antigen binding fragment may comprise a Fab’.

[65] As used herein, the term "single-chain" refers to a molecule comprising amino acid monomers linearly linked by peptide bonds. In certain embodiments, one of the antigen binding moieties, e.g., antigen binding polypeptide construct, is a single-chain Fab molecule, i.e. a Fab molecule wherein the Fab light chain and the Fab heavy chain are connected by a peptide linker to form a single peptide chain. In a particular such embodiment, the C-terminus of the Fab light chain is connected to the N-terminus of the Fab heavy chain in the single- chain Fab molecule. Fv fragments (~25kDa) consist of the two variable domains, VH and VL. Naturally, VH and VL domain are non-covalently associated via hydrophobic interaction and tend to dissociate. However, stable fragments can be engineered by linking the domains with a hydrophilic flexible linker to create a single chain Fv (scFv). In certain other embodiments, one of the antigen binding moieties is a single-chain Fv molecule (scFv).

[66] Single chain fragment variable (scFv) antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In one embodiment, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding. In one embodiment the scFv is a fusion protein comprising the heavy chain variable region and the light chain variable region linked via a peptide linker

[67] The antibody or antigen-binding fragment may be chimeric, human or humanised.

[68] The antibody or antigen-binding fragment may comprise a monoclonal antibody or antigenbinding fragment thereof.

[69] The antibody or antigen-binding fragment may comprise a CH2 domain. The CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody may be the CH2 domain of human IgG 1 , lgG2, lgG3, or lgG4, e.g., the CH2 domain of human IgG 1 . The sequences of human IgG domains are known in the art. [70] The antibody or antigen-binding fragment may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. The hinge region, or part thereof, may be a human lgG1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, may be an lgG1 hinge region, or part thereof.

[71] The sequence of the CH3 domain is not particularly limited. The CH3 domain may be a human immunoglobulin G domain, such as a human IgG 1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[72] The antibody or antigen-binding fragment may comprise a human IgG 1 , lgG2, lgG3, or lgG4 constant region. The sequences of human IgG 1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. The antibody or antigen-binding fragment may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[73] The antibody or antigen-binding fragment may comprise an Fc domain. For example, the first and/or second antibody or antigen-binding fragment may comprise an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise an Fc domain. For example, the first and/or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody and ii) an Fc domain. For example, one of the first or second antibody or antigen-binding fragment may comprise i) a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) and/or a single domain antibody and ii) an Fc domain.

[74] "Fc region", as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences.

[75] The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By "Fc polypeptide" herein is meant one of the polypeptides that make up an Fc region. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as lgG1 , lgG2, lgG3, or lgG4 subtypes, IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprises part or all of a wild type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence. The antibody may comprise a CH2 domain. The CH2 domain is for example located at the N- terminus of the CH3 domain, as in the case in a human IgG molecule. The CH2 domain of the antibody is in one embodiment the CH2 domain of human lgG1 , lgG2, lgG3, or lgG4, e.g the CH2 domain of human lgG1. The sequences of human IgG domains are known in the art.

[76] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise an immunoglobulin hinge region, or part thereof, at the N-terminus of the CH2 domain. The immunoglobulin hinge region allows the two CH2-CH3 domain sequences to associate and form a dimer. In one embodiment, the hinge region, or part thereof, is a human lgG1 , lgG2, lgG3 or lgG4 hinge region, or part thereof. For example, the hinge region, or part thereof, is an IgG 1 hinge region, or part thereof.

[77] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise a CH3 domain. The sequence of the CH3 domain is not particularly limited. In one embodiment, the CH3 domain is a human immunoglobulin G domain, such as a human IgG 1 , lgG2, lgG3, or lgG4 CH3 domain, e.g. a human lgG1 CH3 domain.

[78] Thus, the binding molecule, antibody or antigen binding fragment thereof may comprise a human lgG1 , lgG2, lgG3, or lgG4 constant region. The sequences of human IgG 1 , lgG2, lgG3, or lgG4 CH3 domains are known in the art. A binding molecule, antibody or antigen binding fragment of the invention may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[79] The Fc includes two Fc polypeptides each having a CH3 domain for dimerization. The N- terminal end of each Fc polypeptide is linked to the C-terminus of one of the antigen binding polypeptide constructs with or without a linker.

[80] The Fc region may be any suitable Fc region for example a human Fc region. An Fc region can mediate downstream effector functions via interaction with Fc-receptors found on immune cells or with C1 q, the recognition molecule of the complement system. The antibody or antigenbinding fragment may comprise an Fc region may be capable of interacting with an Fc receptor. The antibody or antigen-binding fragment may comprise an Fc region may be capable of interacting with an Fc receptor and eliciting a downstream effector function. Effector functions refer to downstream immune effector mechanisms such as but not limited to antibody-dependent cytotoxicity, antibody dependent cellular phagocytosis, complement-dependent cytotoxicity, antibody-dependent intracellular neutralization and/or immunomodulatory functions.

[81] Fc receptors (FcRs) are key immune regulatory receptors connecting the antibody mediated (humoral) immune response to cellular effector functions. Receptors for all classes of immunoglobulins have been identified, including FcyR (IgG), FcsRI (IgE), FcaRI (IgA), FcpR (IgM) and FcbR (IgD). There are three classes of receptors for human IgG found on leukocytes: CD64 (FcyRI), CD32 (FcyRlla, FcyRllb and FcyRllc) and CD16 (FcyRllla and FcyRlllb). FcyRI is classed as a high affinity receptor (nanomolar range KD) while FcyRI I and FcyRIII are low to intermediate affinity (micromolar range KD).

[82] The antigen-binding moiety may comprise an additional moiety. The additional moiety may provide further function to the molecule. For example, the further moiety may be selected from a half-life extending moiety and/or a label.

[83] The additional moiety may be selected from one or more half-life extending molecules for example, one or more PEG molecules, a liposome, a serum albumin protein an antibody or antibody fragment that binds serum albumin. The serum albumin may be human serum albumin. The antigen-binding moiety may be conjugated or linked to the additional moiety in any suitable manner. [84] The additional moiety may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

[85] The antibody or antigen-binding fragment may be modified to increase half-life, for example by a chemical modification, especially by PEGylation, or by incorporation in a liposome, or using a serum albumin protein or an antibody or antibody fragment that binds human serum albumin. Increased half-life can also be conferred by conjugating the molecule to an antibody fragment. The term "half-life" as used herein refers to the time taken for the serum concentration of the amino acid sequence, compound or polypeptide to be reduced by 50%, in vivo, for example due to degradation of the sequence or compound and/or clearance or sequestration of the sequence or compound by natural mechanisms.

[86] Increased half-life can also be conferred by conjugating the bispecific molecule to an antibody fragment, for example wherein the antibody fragment binds serum albumin. The antibody fragment that binds serum albumin may be a F(ab')2, Fab, Fab’, Fv, scFv, heavy chain, light chain, variable heavy (VH), variable light (VL) chain, CDR region, single VH or VL domain, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, and bis-scFv, and polypeptides that contain at least a portion of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide. In an embodiment the antigen binding fragment that binds serum albumin may be a single domain antibody for example a VH. In an embodiment the antigen binding fragment that binds serum albumin may be an scFv.

[87] Half-life may be increased by at least 1 .5 times, preferably at least 2 times, such as at least 5 times, for example at least 10 times or more than 20 times, greater than the half-life of the corresponding antibodies without such modification. For example, increased half-life may be more than 1 hours, preferably more than 2 hours, more preferably more than 6 hours, such as more than 12 hours, or even more than 24, 48 or 72 hours, compared to the corresponding antibodies without such modification. The in vivo half-life of the antibody or antigen-binding fragment or compound of the invention a can be determined in any manner known per se, such as by pharmacokinetic analysis. Suitable techniques will be clear to the person skilled in the art. Halflife can for example be expressed using parameters such as the t1 /2-alpha t1/2-beta and the area under the curve (AUC). It will be appreciated by a person skilled in the art that reference to the half-life of an antibody may also refer to the half-life of the compounds of the invention (and may be used interchangeably herein).

[88] In certain embodiments, the antigen-binding moiety may be provided with means to cross the blood-brain barrier. Said means include nanoparticles such as nanoparticles based upon poly(butylcyanoacrylate), poly(lactic-co-glycolic acid), poly(lactic acid), liposomes, and quantum dots. Said nanoparticles preferably are coupled to, for example, transferrin to mediate endocytosis of the coupled nanoparticles by the transferrin receptor at the luminal side, followed by movement through the endothelial cytoplasm and exocytosis at the abluminal (brain) side of the brain capillary endothelium.

[89] The antibody or antigen-binding fragment may be produced by any suitable method. The antibody or antigen-binding fragment may be produced in/by murine, mammal or other animal models, by using hybridoma technology or other methods known in the art.

[90] There are several methods by which to produce recombinant antibodies or antigen-binding fragments thereof which are known in the art. One of these is production in an E. coli expression system. In the E. coli expression system, nucleic acids encoding the antibody or antigen-binding fragment or binding molecule as described herein may be inserted into a plasmid and expressed in a suitable expression system. For example, the present invention includes methods for expressing an antibody or antigen-binding fragment or immunoglobulin chain thereof in a host cell (e.g., bacterial host cell such as E. coli, CHO or other host cell) comprising expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding an immunoglobulin chain that is operably linked to a T7 promoter. For example, a bacterial host cell, such as an E. coli, may include a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside).

[91] Transformation can be by any known method for introducing polynucleotides into a host cell. Methods for introduction of heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide(s) in liposomes, biolistic injection and direct microinjection of the DNA into nuclei. In addition, nucleic acid molecules may be introduced into mammalian cells by viral vectors. Methods of transforming cells are well known in the art.

[92] Thus, the antibody or antigen-binding fragment may be produced by a method comprising the steps of (i) introducing one or more polynucleotides encoding light and/or heavy immunoglobulin domains of the antibody wherein the polynucleotide is in a vector; and/or integrated into a host cell chromosome and/or is operably linked to a promoter; (ii) culturing the host cell (e.g., E. coli, HEK, such as suspension HEK 293 cells, CHO or Pichia or Pichia pastoris) under conditions favourable to expression of the polynucleotide and, (iii) optionally, isolating the antibody or antigen-binding fragment from the host cell and/or medium in which the host cell is grown. When making an antibody or antigen-binding fragment comprising more than one immunoglobulin chain, e.g., an antibody or antigen-binding fragment that comprises two heavy immunoglobulin chains and two light immunoglobulin chains, co-expression of the chains in a single host cell leads to association of the chains, e.g., in the cell or on the cell surface or outside the cell if such chains are secreted, so as to form the antibody or antigen-binding fragment. The methods include those wherein only a heavy immunoglobulin chain or only a light immunoglobulin chain (e.g., any of those discussed herein including mature fragments and/or variable domains thereof) is expressed. Such chains are useful, for example, as intermediates in the expression of an antibody that includes such a chain.

[93] A skilled person will know that there are different ways to identify, obtain and optimise antibodies and antigen-binding fragments that may be used herein, including in vitro and in vivo expression libraries. Optimisation techniques known in the art, such as display (e.g., ribosome and/or phage display) and I or mutagenesis (e.g., error-prone mutagenesis) can be used. The invention therefore also may comprise sequence optimised antibody variants or antigen-binding fragments thereof.

[94] The antigen-binding moiety may comprise a full-length antibody. The full-length antibody may comprise human constant regions and human light chain regions.

[95] According to the first, second, third, fourth and fifth aspects of the invention, the antigenbinding moiety binds to a cell surface receptor that is constitutively internalised and recycled. In such embodiments, the antigen-bonding moiety may bind to any suitable cell surface receptor, i.e., with the proviso that the receptor is constitutively internalised and recycled. By ’’internalising”, “internalised”, etc., and like terms, as used herein is meant a receptor that is capable of being internalised into a cell. By “recycling”, “recycled”, etc., and like terms, as used herein is meant a receptor that, following internalisation, is recycled back to the PM. Receptors that are constitutively internalised and recycled will be known to a person skilled in the art.

[96] The receptor is on the surface of a cell. Thus, by definition, the receptor suitably comprises an extracellular portion. By “extracellular portion”, “extracellular domain”, and like terms as used herein, is meant any portion of a polypeptide chain that is exposed to outside environment of a cell.

[97] It will be appreciated by a person skilled in the art that for a receptor to be constitutively internalised and recycled it should suitably have a cytosolic portion (which can interact with the endomembrane trafficking system, for example). Thus, by definition, the receptor suitably comprises a cytosolic portion. By “cytosolic portion”, “cytosolic domain”, and like terms as used herein, is meant any portion of a polypeptide chain that is exposed to the cytoplasm of a cell.

[98] The receptor may comprise a transmembrane domain (TM). The term “transmembrane domain”, as is used herein, refers to a membrane-spanning protein domain. A transmembrane domain in general comprises non-polar and/or hydrophobic amino acid residues. The presence of a transmembrane domain can be predicted using, for example, hydrophobicity analysis. Computational resources are available, such as HMMTOP, TMpred, TMHMM and TopPred2, that may help in predicting transmembrane domains in protein sequences.

[99] The receptor may suitably comprise each of an extracellular portion, a transmembrane domain and a cytosolic portion.

[100] The receptor may comprise a multi-domain protein and/or a protein complex. By “multidomain protein” as used herein is meant a protein wherein two or more domains are found in a single polypeptide chain. By “protein complex” as used herein is meant a group of two or more associated polypeptide chains. Thus, in certain embodiments, the receptor may comprise each of an extracellular portion, a transmembrane domain and a cytosolic portion in the form of a multidomain protein. In certain alternative embodiments, the receptor may comprise each of an extracellular portion, a transmembrane domain and a cytosolic portion in the form of a protein complex.

[101] Preferably, the receptor comprises each of an extracellular portion, a transmembrane domain and a cytosolic portion in the form of a multi-domain protein.

[102] Internalisation from the PM is most commonly effected by clathrin-mediated endocytosis. There are three predominant types of motifs that mark receptor proteins for clathrin-mediated endocytosis:

• NXXY, such as NPXY, wherein X designates any amino acid residue;

• [D/E/Q/N/K/R/H]XXXL[L/I/V/M], such as [D/E]XXXL[L/I], wherein 7’ designates ‘or’ and X designates any amino acid residue: these motifs are often called 'acidic dileucine' or simply 'dileucine' motifs;

• [Y/W]XX[F/L/I/M/V], such as YXX[F/L/I/M/V], wherein 7’ designates ‘or’ and X designates any amino acid residue: these motifs are often called simply 'tyrosine motifs'.

[103] NXXY, such as NPXY motifs may be bound by the clathrin adaptors Dab2, ARH and NUMB, for example.

[104] [D/E/Q/N/K/R/H]XXXL[L/I/V/M], such as [D/E]XXXL[L/I], motifs may be bound by the 'sigma/alpha' subunits of the AP2 clathrin adaptor complex, for example. There are also variant dileucine motifs that have been shown to interact with AP2, for example, to direct TM protein internalisation.

[105] [Y/W]XX[F/L/I/M/V], such as YXX[F/L/I/M/V] motifs may be bound by the 'mu' subunit of the AP2 clathrin adaptor complex, for example.

[106] A further type of internalisation signal is KXXM, wherein X designates any amino acid residue. These motifs are present in TM proteins called R-SNAREs, such as VAMP8, for example. R-SNAREs have folded, structural motifs that may be recognized by the clathrin adaptor CALM (VAMP8), for example, instead of linear, unstructured sequence motifs. The motif, i.e., KXXM, is typically present on the face of an accessible helix (i.e., not structurally buried) of a receptor protein, such as a TM protein.

[107] In other cases, TM proteins that lack such motifs might be internalised by clathrin-mediated endocytosis, for instance by association with another TM protein, such as in the case of PD-L1 , for example, and/or association with a cytosolic protein that has such a motif, such as in the case of the HIV Nef protein, for example.

[108] Recycling of TM proteins may be effected by recycling motifs. Exemplary recycling motifs include NXXY, such as NPXY, wherein X represents any amino acid residue. Another exemplary recycling motif is X⁵XX⁶, wherein X represents any amino acid residue; X⁵ represents F, W, Y or H; and X⁶ represents L or M.

[109] It will be appreciated that although reference herein is made to “internalisation and/or recycling” motifs, a motif, such as any of those defined herein (or others), may be either an internalisation motif or a recycling motif, or may be both an internalisation motif and a recycling motif. For example, and by way of example only, a person skilled in the art would understand the following to be internalisation motifs:

• X¹XXX², wherein X represents any amino acid residue; X¹ represents Y or W, such as Y; and X² represents L, I, V, M or F;

• X³XXXLX⁴, wherein X represents any amino acid residue; X³ represents D, E, Q, N, K, R, or H, such as such as D or E; and X⁴ represents L, I, V or M, such as L or I; and

• KXXM, wherein X represents any amino acid residue.

[110] For example, and by way of example only, a person skilled in the art would understand the following to be recycling motifs:

• X⁵XX⁶, wherein X represents any amino acid residue; X⁵ represents F, W, Y or H; and X⁶ represents L or M.

[111] For example, and by way of example only, a person skilled in the art would understand the following to be internalisation and recycling motifs:

• NXXY, such as NPXY, wherein X represents any amino acid residue.

[112] The antigen-binding moiety may be operable to bind to a receptor having an X¹XXX² internalisation and/or recycling motif, wherein X represents any amino acid residue; X¹ represents Y or W, such as Y; and X² represents L, I, V, M or F.

[113] The antigen-binding moiety may be operable to bind to a receptor having an X³XXXLX⁴ internalisation and/or recycling motif, wherein X represents any amino acid residue; X³ represents D, E, Q, N, K, R, or H, such as such as D or E; and X⁴ represents L, I, V or M, such as L or I.

[114] The antigen-binding moiety may be operable to bind to a receptor having a KXXM internalisation and/or recycling motif, wherein X represents any amino acid residue.

[115] The antigen-binding moiety may be operable to bind to a receptor having an NXXY internalisation and/or recycling motif, such as NPXY, wherein X represents any amino acid residue.

[116] The antigen-binding moiety may be operable to bind to a receptor having an X⁵XX⁶ internalisation and/or recycling motif, wherein X represents any amino acid residue; X⁵ represents F, W, Y or H; and X⁶ represents L or M.

[117] The internalisation and/or recycling motif may be other than KXXM and, in such embodiments, may be located in an unstructured region of the receptor; or wherein the internalisation and/or recycling motif is KXXM and is located in an accessible helix of the receptor, wherein X represents any amino acid residue.

[118] In some embodiments, the receptor may comprise only an internalisation motif.

[119] In some embodiments, the receptor may comprise only a recycling motif.

[120] In some embodiments, the receptor may comprise an internalisation motif and a recycling motif.

[121] As defined herein, the receptor suitably comprises a cytosolic portion. The internalisation and/or recycling motif may be located in the cytosolic portion of the receptor. By “cytosolic” is meant located on the interior of the cell on the inner side of the plasma membrane. Preferably, the internalisation and/or recycling motif may be located in an accessible segment of the cytosolic portion of the receptor. The internalisation and/or recycling motif(s) may be accessible by virtue of being in an unstructured part of the cytosolic portion of the receptor, for example. For example, the internalisation and/or recycling motif may be located at least 5 residues, such as at least 6 residues, away from the predicted start of the cytosolic portion (or domain).

[122] The internalisation and/or recycling motif may not be YQRL.

[123] Examples of internalisation and/or recycling motifs are provided, by way of example only, in Table 1 below.

Table 1 : Example internalisation and/or recycling motifs

[124] As discussed herein, receptors that are constitutively internalised and recycled will be known to a person skilled in the art. The ability of a receptor to undergo constitutive internalisation and recycling may be predicted based on the presence of one or more of the internalisation and/or recycling motif defined herein, suitably based on the presence of one or more of the internalisation and/or recycling motif defined herein in a cytosolic portion of the receptor. It will be appreciated by a person skilled in the art that the internalisation and/or recycling motif(s) provided herein should be present in a cytosolic portion of a suitable receptor in order for said motif(s) to drive internalisation and/or recycling. Further, said motif(s) should suitably be accessible. In other words, the presence of one or more of the internalisation and/or recycling motif(s) defined herein may not be indicative of internalisation and/or recycling alone, rather said motif(s) should also be present in a cytosolic portion of the receptor and, preferably, be accessible (to the endosomal trafficking system, for example). As defined hereinabove, the internalisation and/or recycling motif(s) may be accessible by virtue of being in an unstructured part of the cytosolic portion of the receptor, for example.

[125] The antigen-binding moiety may bind to one or more receptor(s) selected from the group consisting of: HCMV US28; Erbb2; CAIX; p-glycoprotein; ADAMs, such as ADAM17; integrins; CDs, such as CD22; CD33, CD56, CD70 and CD71 ; GLUT1 ; GPCRs; LDLR; PD-1 ; PD-L1 ; voltage-gated sodium channels, such as Nav1.1 , Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Na_v1.7, Na_v1.8 and Na_v1.9; TRKA/NTRK1 channels; R-SNAREs, such as VAMP2, VAMP3, VAMP4, VAMP7 and VAMP8; LAMP1 ; LAMP2; HER2; HER3; CTLA-4, Frizzled receptors, such as Frizzled-1 , Frizzled-2, Frizzled-3, Frizzled-4, Frizzled-5, Frizzled-6, Frizzled-7, Frizzled-8, Frizzled-9 and Frizzled-10; IL1 RAP; IL2RA; IL2RB; BACE1 ; BACE2; amyloid beta precursor protein; APLP1 ; APLP2; subunits of the gamma secretase complex, such as APH1A, APH1 B, Nicastrin, Presenilin and PENSEN; TGFBR1 ; EPHB2; OSMR; MUC1 ; NRP1 ; ROR1 ; ICAM1 ; EPHA3; SEMA4B; GPR107; and VEGFR2. It will be appreciated by a person skilled in the art that each of these receptors are constitutively internalised and/or recycled. It will also be appreciated by a person skilled in the art that each of these receptors i) comprise one or more of the internalisation and/or recycling motifs defined herein and/or ii) have been shown experimentally to be internalising and/or recycling.

[126] Preferably, the antigen-binding moiety may bind to US28.

[127] The term US28 refers to a G-protein coupled receptor that is encoded by herpesviruses, especially CMV. US28 is a rare multi-chemokine family binding receptor with the ability to bind ligands such as CCL2/MCP-1 , CCL5/RANTES, and CX3CLI/fraktalkine as ligands. Ligand binding to US28 activates cell-type and ligand-specific signalling pathways leading to cellular migration, which is an important example of receptor functional selectivity. Additionally, US28 has been demonstrated to constitutively activate Gaq, phospholipase C (PLC) and NF-kB signaling pathways, amongst others. The term CMV refers to a virus of the genus Cytomegalovirus, which currently harbours eight species. Human cytomegalovirus (HCMV), also termed human herpesvirus 5 (HHV-5) is the type species.

[128] Examples of suitable antigen-binding moieties which bind US28 are described in the published patent application WO 2019/151865 A1 , the full contents of which are incorporated herein by reference.

[129] The antigen-binding moiety may comprise an anti-US28 antibody or fragment thereof, such as an antibody or fragment thereof comprising the amino acid sequence as set forth in SEQ ID NO. 6. In some embodiments, the antigen-binding moiety may comprise two or more copies of the amino acid sequence as set forth in SEQ ID NO. 6, for example linked by a suitable peptide linker (such as the Gly-Ser linkers described hereinbelow). For example, the antigen-binding moiety may comprise an anti-US28 antibody as set forth in SEQ ID NO. 7.

[130] The antigen-binding moiety may comprise an anti-CD71 antibody or fragment thereof, such as an antibody or fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO. 8 and/or SEQ ID NO. 9, preferably as set forth in SEQ ID NO. 8. For example, the antigenbinding moiety may comprise OKT9. The antigen-binding moiety may comprise a humanized version of the amino acid sequences as set forth in SEQ ID NO. 8 and/or SEQ ID NO. 9, preferably a humanized version of the amino acid sequence as set forth in SEQ ID NO. 8.

[131] The antigen-binding moiety may comprise an anti-HER2 antibody or fragment thereof, such as an antibody or fragment thereof comprising the amino acid sequences as set forth in SEQ ID NO. 10 and/or SEQ ID NO. 11 , preferably as set forth in SEQ ID NO. 10. For example, the antigenbinding moiety may comprise Trastuzumab.

[132] As such, the antigen-binding moiety may comprise an amino acid sequence selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11 .

[133] The conjugate comprises the C-terminal domain of PCSK9 or a homolog thereof.

[134] Human PCSK9 (SEQ ID NO. 12) has 692 amino acids and contains a pro-domain (residues 31-152); a catalytic domain (residues 153-451); and a C-terminal domain (residues 452-692), which is further divided into three modules (Du F, Hui Y, Zhang M, Linton MF, Fazio S, Fan D (December 2011). "Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein". Journal of Biological Chemistry. 286 (50): 43054-43061). The N-terminal pro-domain has a flexible crystal structure and is responsible for regulating PCSK9 function by interacting with and blocking the catalytic domain, which otherwise binds the epidermal growth factor-like repeat A (EGF-A) domain of the LDLR (Du F, Hui Y, Zhang M, Linton MF, Fazio S, Fan D (December 2011). "Novel domain interaction regulates secretion of proprotein convertase subtilisin/kexin type 9 (PCSK9) protein". Journal of Biological Chemistry. 286 (50): 43054-43061 , Lo Surdo P, Bottomley MJ, Calzetta A, Settembre EC, Cirillo A, Pandit S, et al. (December 2011). "Mechanistic implications for LDL receptor degradation from the PCSK9/LDLR structure at neutral pH". EMBO Reports. 12 (12): 1300-1305 and Piper DE, Jackson S, Liu Q, Romanow WG, Shetterly S, Thibault ST, et al. (May 2007). "The crystal structure of PCSK9: a regulator of plasma LDL-cholesterol". Structure. 15 (5): 545-552).

[135] The C-terminal domain of PCSK9 may comprise or consist of amino acid residues 450 to 692 of human PCSK9 as set forth in SEQ ID NO. 1 .

[136] The C-terminal domain of PCSK9 may comprise or consist of amino acid residues 452 to 692 of human PCSK9 as set forth in SEQ ID NO. 2.

[137] The C-terminal domain of PCSK9 may comprise or consist of amino acid residues 450 to 529 of human PCSK9 as set forth in SEQ ID NO. 3.

[138] The C-terminal domain of PCSK9 may comprise or consist of amino acid residues 450 to 605 of human PCSK9 as set forth in SEQ ID NO. 4.

[139] In humans, PCSK9 is encoded by the PCSK9 gene. Similar genes (homologs, orthologs, paralogs) are found across many species.

[140] Thus, while aspects and embodiments relating to human PCSK9 herein typically refer to the protein according to its designation in humans, for cells of another family or species, it is within the level of skill in the art to identify the corresponding gene or protein, e.g. , the homolog, ortholog and/or paralog, in the other family or species, typically by identifying sequences having moderate (typically >30%) or high (typically >50%) identity to the human sequence, preferably taking the function of the protein expressed by the gene and/or the locus of the gene in the genome into account.

[141] A skilled person would thus understand that the various aspects of the invention extend to mutant proteins and to PCSK9 proteins in other species. As used herein, a homolog of a nucleic acid sequence or amino acid sequence has at least 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 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%, 77%, 78%, 79%,

80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%,

96%, 97%, 98%, or 99% overall sequence identity to the wild type nucleic acid or amino acid sequence.

[142] "Identity" is a property of sequences that measures their similarity or relationship. The term "sequence identity" or "identity" as used in the present disclosure means the percentage of pair- wise identical residues - following (homologous) alignment of a sequence of a polypeptide of the disclosure with a sequence in question - with respect to the number of residues in the longer of these two sequences. Sequence identity is measured by dividing the number of identical amino acid residues by the total number of residues and multiplying the product by 100.

[143] The term "homology" is used herein in its usual meaning and includes identical amino acids as well as amino acids which are regarded to be conservative substitutions (for example, exchange of a glutamate residue by an aspartate residue) at equivalent positions in the linear amino acid sequence of a polypeptide of the disclosure.

[144] Suitable homologs or orthologs can be identified by sequence comparisons and identifications of conserved domains using databases such as NCBI and Paint ensemble and alignment programmes known to the skilled person. The percentage of sequence homology or sequence identity can, for example, be determined herein using the program BLASTP.

[145] Alignments can be carried out using suitable computer programs such as BLAST2.0, which stands for Basic Local Alignment Search Tool or ClustalW or any other suitable program which is suitable to generate sequence alignments. Accordingly, a wild-type nucleic acid or amino acid sequence can serve as "subject sequence" or "reference sequence", while the amino acid sequence of a mutant nucleic acid or amino acid sequence different from the wild-type described herein serves as "query sequence". The terms "reference sequence" and "wild- type sequence" are used interchangeably herein.

[146] For the purposes of the invention, a "mutant" protein is a protein that has been altered compared to a naturally occurring wild type (WT) protein. For example, a mutant human PCSK9 protein is one that has been altered compared to the naturally occurring wild type (WT) human PCSK9 protein. [147] A mutation in a nucleic acid sequence or amino acid sequence can be a deletion, insertion or substitution of one or more residue. Mutations in a nucleic acid sequence can lead to a missense mutation resulting in the substitution of a single amino acid in the protein. Alternatively, a mutation in a nucleic acid sequence can introduce a premature stop codon resulting in a truncated protein or a change in the subsequent amino acid sequence. A knock out mutation is a mutation that abolishes function of the protein.

[148] In some embodiments, the C-terminal domain of PCSK9 may comprise a mutant C-terminal domain of human PCSK9, such as a domain comprising a substitution of any one or more of residues 549-558 of the C-terminal domain of human PCSK9, such as a domain comprising a substitution of any one or more of residues 549-558 of the C-terminal domain of human PCSK9 to provide residues 549-558 with an overall positive charge, such as a domain comprising a substitution of any one or more of residues 549-558 of the C-terminal domain of human PCSK9 to lysine (K), arginine (R) and/or histidine (H), such as a domain having a H553R substitution and/or a Q554R substitution, preferably a H553R substitution and a Q554R substitution, such as that set forth in SEQ ID NO. 5.

[149] In some embodiments, the C-terminal domain of PCSK9 may comprise a mutant C-terminal domain of human PCSK9, such as a domain comprising a substitution of any one or more of residues 453-529 of the C-terminal domain of human PCSK9, such as a domain comprising a substitution of any one or more of residues 453-529 of the C-terminal domain of human PCSK9 to serine (S), such as a domain having a W453S substitution, a L455S substitution and/or a L529S substitution, such as that set forth in SEQ ID NO. 36.

[150] In some embodiments, the C-terminal domain of PCSK9 may comprise a mutant C-terminal domain of human PCSK9, such as a domain comprising a deletion of any one or more of residues 532-692 of the C-terminal domain of human PCSK9, such as a domain comprising a deletion of residues 532-603 and/or residues 604-692 of the C-terminal domain of human PCSK9, such as that set forth in SEQ ID NO. 39 and/or SEQ ID NO. 4, respectively.

[151] The conjugate may comprise a linker. Preferably, the linker may join the antigen-binding moiety and the C-terminal domain of PCSK9. When a linker is present, the conjugate may be of the following formula: A-L-P, wherein A represents an antigen-binding moiety, L represents a bivalent linker, and P represents the C-terminal domain of PCSK9.

[152] The linker may comprise any suitable linker.

[153] Preferably, the linker may comprise a polypeptide. For example, the linker may comprise a polypeptide comprising from 1 to 60 amino acid residues, preferably from 10 to 40 amino acid residues, most preferably 35 amino acid residues, such as 30 amino acid residues, 31 amino acid residues, 32 amino acid residues, 33 amino acid residues, 34 amino acid residues, 35 amino acid residues, 36 amino acid residues, 37 amino acid residues, 38 amino acid residues, or 39 amino acid residues.

[154] The linker may comprise a poly-alanine linker, such as AAA. [155] The linker may comprise a Gly-Ser linker (otherwise known as a GS-linker; these terms may be used interchangeably herein). For example, the linker may comprise the sequence (Gly_xSer_y)z, wherein x may be from 1 to 10, such as from 1 to 5, such as from 2 to 5, preferably 3 or 4; y may be from 1 to 5, such as from 1 to 4, such as from 1 to 3, preferably 1 or 2; and z may be from 1 to 20, such as from 1 to 10, such as from 2 to 10, such as from 3 to 8, preferably from 3 to 7.

[156] Examples of suitable Gly-Ser linkers include, but are not limited to, one or more of the following: (GGGGS)s, (GGGGS)? and/or (GGGSS)s.

[157] Preferably, the linker may comprise the sequence (GGGS)_n, wherein n is from 1 to 20, such as from 1 to 10.

[158] The Gly-Ser linker may comprise

GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (“GS30”; SEQ ID NO. 13) or GGGGSGGGS ("GS9"; SEQ ID NO. 14).

[159] Examples of suitable Gly-Ser linkers are also described in the published patent applications WO 99/42077, WO 06/040153 and WO 06/122825, the full contents of which are each fully incorporated herein. WO 06/040153 and WO 06/122825 describe GS30, GS15, GS9 and GS7 linkers, for example.

[160] The linker may comprise a hinge-like region, such as the hinge regions of naturally occurring heavy chain antibodies or similar sequences. Examples of suitable hinge-like regions are also described in the published patent application WO 94/04678, the full contents of which is fully incorporated herein.

[161] Other suitable linkers may comprise organic compounds or polymers, in particular those suitable for use in proteins for pharmaceutical use. For instance, poly(ethyleneglycol) moieties have been used to link antibody domains. Examples of such linkers are described in the published patent application WO 04/081026, the full contents of which is fully incorporated herein.

[162] The conjugate may be produced by any suitable method. In some embodiments, the conjugate may be produced recombinantly.

[163] Suitable recombinant techniques will be known to a person skilled in the art. For example, nucleic acids encoding the antigen-binding moiety, the C-terminal domain of PCSK9 or a homolog thereof and optionally a peptide linker, may be inserted into a plasmid and expressed in a suitable expression system. For example, nucleic acids encoding A-L_n-P, wherein A represents the antigen-binding moiety, L represents a peptide linker, n is 0 or 1 , and P represents the C-terminal domain of PCSK, may be expressed in a suitable expression system. Suitable expression systems include bacterial host cells, such as E. coli, CHO or other host cells expressing T7 RNA polymerase in the cell which also includes a polynucleotide encoding A-L_n-P that is operably linked to a T7 promoter. For example, a bacterial host cell, such as an E. coli, may include a polynucleotide encoding the T7 RNA polymerase gene operably linked to a lac promoter and expression of the polymerase and the A-Ln-P chain is induced by incubation of the host cell with IPTG (isopropyl-beta-D-thiogalactopyranoside). Transformation can be by any known method for introducing polynucleotides into a host cell. Suitable transformation methods are as already described herein.

[164] In some embodiments, the conjugate may further comprise a payload.

[165] A skilled person would know that different payloads are known in the art (see e.g., Gingrich J. How the Next Generation Antibody Drug Conjugates Expands Beyond Cytotoxic Payloads for Cancer Therapy - J. ADC. April 7, 2020).

[166] In one embodiment, the payload may be a cell killing agent.

[167] In one embodiment, the payload may be a macrophage class switching agent.

[168] In one embodiment, the payload may be an immune-modulating payload.

[169] In one embodiment, the payload may be a light activatable payload.

[170] In one embodiment, the payload may be a molecular label. By “molecular label”, and like terms as used herein, is meant a group that is operable to aid the detection of the compound. Detection of the compound may be ex vivo and/or in vivo. Examples of suitable molecular labels include, but are not limited to, fluorescent molecules, p-galactosidase, luciferase molecules, chemical dyes, fluorophores and/or radioisotopes.

[171] The payload may be a detectable or functional label. A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorophores, fluorescers, radiolabels, enzymes, chemiluminescers, a nuclear magnetic resonance active label or photosensitizers. Thus, the binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance. The molecular label may be a fluorophore. Suitable fluorophores include fluorescein isothiocyanate (FITC), phycoerythrin (PE), allophycocyanin (APC), Indocicarbocyanine (Cy5), Indocarbocyanine (Cy3), as well as those known by the trade names Alexa Fluor (such as 350, 405, 488, 532, 546, 568, 594, 647, 680, 700, 750) and DyLight (such as 405, 488, 550, 650, 680, 755, 800).

[172] The molecular label may also be a biotin tag, derived from biotin. The labelling moiety may also be a radioisotope or a radioisotope containing moiety. Suitably, the labelling moiety is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [1 1 C] methionine, [1 1 C] choline, copper Cu dotatate, [18F] EF5, [18F] fluciclovine, [18F] fluorocholine, [18F] fluoroethyl- L-tyrosine, [18F] fluoromisonidazole, [18F] fluorothymidine F-18, [64 Cu] Cu-ETS2, [68Ga] DOTA- pseudopeptides, [68Ga] DOTA-TATE and [68Ga] prostate-specific membrane antigen (PSMA).

[173] As used herein, an immune-modulating payload includes any moiety that modulates the immune system, for example which stimulates the immune system and/or kills the target cell. Thus, a moiety that has immuno-activating and/or antineoplastic activities can be used. Such moieties may be synthetic peptides that recognise the specific target and trigger (agonist) or block (antagonist) inflammatory responses. The target may be a pattern recognition receptor (PRR), including Toll-like receptors (TLRs), NOD-like receptors (NLRs), RIG-l-like receptors (RLRs), C- type lectin receptors (CLRs) and cytosolic dsDNA sensors (CDSs). [174] Examples of payloads include agonists for the stimulator of interferon genes protein (STING; transmembrane protein 173; TMEM173). Such payloads include cyclic dinucleotides and compounds listed in see WO2021113679). Activation of the STING pathway triggers an immune response that results in generation of specific killer T-cells that shrink tumours and can provide long-lasting immunity so the tumours do not recur. Alternatively, payloads that act on toll-like receptors (TLRs) may be used. For example, agonists that bind to TLR7 and/or TLR8 can be used.

[175] Another example is a macrophage class switching agent.

[176] A light activatable payload (IRDye® 700DX, IR700) may also be used. Light activation of the non-toxic payload results in the generation of singlet oxygen species that damage the cell membrane integrity, resulting in necrotic and immunogenic cell death of tumour cells, resulting in minimal damage to surrounding normal tissue.

[177] The cell killing agent may be a cytotoxin and the skilled person will understand that a range of cytotoxins will be compatible. For example, suitable cytotoxins include, but are not limited to: i) a peptide toxin, or ii) a chemical toxin, or iii) an inhibitor of Bcl-2 or Bcl-axl, iv) an RNA Polymerase inhibitor such as a-amanitin, v) a spliceosome inhibitor, vi) a microtubule-targeting payload, or vii) a DNA-damaging payload.

[178] The payload may be a cytotoxic payload or a therapeutic compound, peptide or polypeptide. In particular, the payload is preferably a cytotoxin. The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes (e.g., <211 >At, <131 >l, <125>1 , <90>Y, <186>Re, <188>Re, <153>Sm, <212>Bi, <32>P, <60>C, and radioactive isotopes of Lu), chemotherapeutic agents, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including synthetic analogs and derivatives thereof.A "chemotherapeutic agent" and "anticancer agent" are terms that denote a chemical compound useful in the treatment of cancer, and which may be administered in combination therapy with the antibody drug conjugate compounds of the invention. Examples of chemotherapeutic agents include Erlotinib (TARCEV A(R), Genentech/OSI Pharm.), Bortezomib (VELCADE(R), Millenium Pharm.), Fulvestrant (FASLODEX(R), Astrazeneca), Sutent (SU1 1248, Pfizer), Letrozole (FEMARA(R), Novartis), Imatinib mesylate (GLEEVEC(R), Novartis), PTK787/ZK 222584 (Novartis), Oxaliplatin (Eloxatin(R), Sanofi), 5-FU (5-fluorouracil), Leucovorin, Rapamycin (Sirolimus, RAPAMUNE(R), Wyeth), Lapatinib (GSK572016, GlaxoSmithKline), Lonafarnib (SCH 66336), Sorafenib (BAY43-9006, Bayer Labs.), and Gefitinib (IRESSA(R), Astrazeneca), AG1478, AG1571 (SU 5271 ; Sugen), alkylating agents such as thiotepa and CYTOXAN(R) cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylolomelamine; TLK 286 (TELCYTA(TM)); acetogenins (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinol (dronabinol, MARINOL(R)); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN(R)), CPT-II (irinotecan, CAMPTOSAR(R)), acetylcamptothecin, scopolectin, and 9- aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; bisphosphonates, such as clodronate; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammall and calicheamicin omegall (see, e.g., Agnew, Chem Inti. Ed. Engl, 33: 183-186 (1994)) and anthracyclines such as annamycin, AD 32, alcarubicin, daunorubicin, dexrazoxane, DX-52-1 , epirubicin, GPX- 100, idarubicin, KRN5500, menogaril, dynemicin, including dynemicin A, an esperamicin, neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores, aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin, detorubicin, 6-diazo-5-oxo- L-norleucine, ADRIAMYCIN(R) doxorubicin (including morpholino- doxorubicin, cyanomo[phi]holino-doxorubicin, 2-pyrrolino-doxorubicin, liposomal doxorubicin, and deoxydoxorubicin), esorubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, and zorubicin; folic acid analogues such as denopterin, pteropterin, and trimetrexate; purine analogs such as fludarabine, 6- mercaptopurine, thiamiprine, and thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, and floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, and testolactone; anti-adrenals such as aminoglutethimide, mitotane, and trilostane; folic acid replenisher such as folinic acid (leucovorin); aceglatone; anti-folate anti-neoplastic agents such as ALEMTA(R), LY231514 pemetrexed, dihydrofolate reductase inhibitors such as methotrexate, antimetabolites such as 5-fluorouracil (5-FU) and its prodrugs such as UFT, S-l and capecitabine, and thymidylate synthase inhibitors and glycinamide ribonucleotide formyltransferase inhibitors such as raltitrexed (TOMUDEX<1 >^A, TDX); inhibitors of dihydropyrimidine dehydrogenase such as eniluracil; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISENE(R), FILDESIN (R)); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara- C"); cyclophosphamide; thiotepa; taxoids and taxanes, e.g., TAXOL(R) paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ.), ABRAXANE(TM) Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE(R) doxetaxel (Rh[delta]ne-Poulenc Rorer, Antony, France); chloranbucil; gemcitabine (GEMZAR(R)); 6-thioguanine; mercaptopurine; platinum; platinum analogs or platinum-based analogs such as cisplatin, oxaliplatin and carboplatin; vinblastine (VELBAN(R)); etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN(R)); vinca alkaloid; vinorelbine (NAVELBINE(R)); novantrone; edatrexate; daunomycin; aminopterin; xeloda; ibandronate; topoisomerase inhibitor RFS 2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid; pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATESf(TM)) combined with 5-FU and leucovorin.

[179] Also encompassed by the term “cytotoxic agent” are anti-hormonal agents that act to regulate or inhibit hormone action on tumors such as anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX(R) tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYI 17018, onapristone, and FARESTON(R) toremifene; aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)- imidazoles, aminoglutethimide, MEGASE(R) megestrol acetate, AROMASIN(R) exemestane, formestanie, fadrozole, RIVISOR(R) vorozole, FEMARA(R) letrozole, and ARHVIIDEX(R) anastrozole; and anti-androgens such as fiutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as well as troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as gene therapy vaccines, for example, ALLOVECTIN(R) vaccine, LEUVECTIN(R) vaccine, and VAXID(R) vaccine; PROLEUKIN(R) rlL- 2; LURTOTECAN(R) topoisomerase 1 inhibitor; ABARELIX(R) rmRH; and pharmaceutically acceptable salts, acids or derivatives of any of the above

[180] Preferably the cytotoxin is a biologically active cytotoxic material. The cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camptothecin analogues and doxorubicin.

[181] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials. [182] In some embodiments, the conjugate may further comprise (or is incorporated or associated with) a cytotoxic or cytostatic agent, i.e., a compound that kills or inhibits cells such as tumour cells. Such agents may impart their cytotoxic and cytostatic effects by mechanisms including tubulin binding, DNA binding, proteasome and/or topoisomerase inhibition.

[183] The cytotoxic or cytostatic agent may be, for example, a peptide toxin, a small molecule toxin or a radioisotope. This is also referred to herein as drug or cytotoxic payload.

[184] As used herein, an “ADC” is an antibody drug conjugate.

[185] In one embodiment the cytotoxic or cytostatic agent may be a tubulin inhibitor; or a DNA interacting agent. Tubulin inhibitors modulate tubulin polymerisation. DNA interacting agents target cellular DNA.

[186] In an embodiment the cytotoxic or cytostatic agent is a tubulin inhibitor. In an embodiment, the tubulin inhibitor is selected from the group consisting of: (a) an auristatin; and (b) a maytansine derivative. In an embodiment, the cytotoxic or cytostatic agent is an auristatin. Auristatins include synthetic derivatives of the naturally occurring compound Dolastatin-10. Auristatins are a family of antineoplastic I cytostatic pseudopeptides. Dolastatins are structurally unique due to the incorporation of 4 unusual amino acids (Dolavaine, Dolaisoleuine, Dolaproine and Dolaphenine) identified in the natural biosynthetic product. In addition, this class of natural product has numerous asymmetric centres defined by total synthesis studies by Pettit et al (US 4,978,744). It would appear from structure activity relationships that the Dolaisoleuine and Dolaproine residues appear necessary for antineoplastic activity (US 5,635,483 and US 5,780,588). In an embodiment, the auristatin is selected from the group consisting of: Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF); vcMMAE; vcMMAF; mcMMAE and mcMMAF. In an embodiment, the cytotoxic or cytostatic agent is a maytansine or a structural analogue of maytansine. In an embodiment, the cytotoxic or cytostatic agent is a maytansine. Maytansines include structurally complex antimitotic polypeptides. Maytansines are potent inhibitors of microtubulin assembly which leads towards apoptosis of tumour cells. In an embodiment the maytansine is selected from the group consisting of: Mertansine (DM1); and a structural analogue of maytansine such as DM3 or DM4. Preferably, the drug is MMAE, MMAF or auristatin MMAF.

[187] In an embodiment, the cytotoxic or cytostatic agent is DNA interacting agent. In an embodiment, the DNA interacting agent is selected from the group consisting of: (a) calicheamicins, (b) duocarmycins and (c) pyrrolobenzodiazepines (PBDs). In an embodiment, the cytotoxic or cytostatic agent is a calicheamicin. Calicheamicin is a potent cytotoxic agent that causes double-strand DNA breaks, resulting in cell death. Calicheamicin is a naturally occurring enediyne antibiotic (A. L. Smith et al, J. Med. Chem., 1996, 39,11 , 2103-21 17). Calicheamicin was found in the soil microorganism Micromonosporaechinospora. In an embodiment, the calicheamicin is calicheamicin gamma 1. In an embodiment, the drug is a duocarmycin. Duocarmycins are potent anti-tumour antibiotics that exert their biological effects through binding sequence-selectively in the minor groove of DNA duplex and alkylating the N3 of adenine (D. Boger, Pure & Appl. Chem., 1994, 66, 4, 837-844). In an embodiment, the duocarmycin is selected from the group consisting of: Duocarmycin A; Duocarmycin B1 ; Duocarmycin B2; Duocarmycin C1 ; Duocarmycin C2; Duocarmycin D; Duocarmycin SA; Cyclopropylbenzoindole (CBI) duocarmycin; Centanamycin; Rachelmycin (CC-1065); Adozelesin; Bizelesin; and Carzelesin. In an embodiment, the cytotoxic or cytostatic agent is a pyrrolobenzodiazepine. Pyrrolobenzodiazepines (PBDs) are a class of naturally occurring anti-tumour antibiotics. Pyrrolobenzodiazepines are found in Streptomyces. PBDs exert their anti-tumour activity by covalently binding to the DNA in the minor groove specifically at purine-guanine-purine units. They insert on to the N2 of guanine via an aminal linkage and, due to their shape, they cause minimal disruption to the DNA helix. It is believed that the formation of the DNA-PBD adduct inhibits nucleic acid synthesis and causes excision-dependent single and double stranded breaks in the DNA helix. As synthetic derivatives the joining of two PBD units together via a flexible polymethylene tether allows the PBD dimers to cross-link opposing DNA strands producing highly lethal lesions. In an embodiment, the cytotoxic or cytostatic agent is a synthetic derivative of two pyrrolobenzodiazepines units joined together via a flexible polymethylene tether. In an embodiment, the pyrrolobenzodiazepine is selected from the group consisting of: Anthramycin (and dimers thereof); Mazethramycin (and dimers thereof); Tomaymycin (and dimers thereof); Prothracarcin (and dimers thereof); Chicamycin (and dimers thereof); Neothramycin A (and dimers thereof); Neothramycin B (and dimers thereof); DC-81 (and dimers thereof); Sibiromycin (and dimers thereof); Porothramycin A (and dimers thereof); Porothramycin B (and dimers thereof); Sibanomycin (and dimers thereof); Abbeymycin (and dimers thereof); SG2000; and SG2285.

[188] In an embodiment, the cytotoxic or cytostatic agent is a drug that targets DNA interstrand crosslinks through alkylation. A drug that targets DNA interstrand crosslinks through alkylation is selected from: a DNA targeted mustard; a guanine-specific alkylating agent; and an adeninespecific alkylating agent. In an embodiment, the cytotoxic or cytostatic agent is a DNA targeted mustard. For example, the DNA targeted mustard may be selected from the group consisting of: an oligopyrrole; an oligoimidazole; a Bis-(benzimidazole) carrier; a Polybenzamide Carrier; and a 9-Anilinoacridine-4-carboxamide carrier.

[189] In an embodiment, the cytotoxic or cytostatic agent is selected from the group consisting of: Netropsin; Distamycin; Lexitropsin; Tallimustine; Dibromotallimustine; PNU 157977; and MEN 10710.

[190] In an embodiment, the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258.

[191] A guanine-specific alkylating agent is a highly regiospecific alkylating agents that reacts at specific nucleoside positions. In an embodiment, the cytotoxic or cytostatic agent is a guaninespecific alkylating agent selected from the group consisting of: a G-N2 alkylators; a A-N3 alkylator; a mitomycin; a carmethizole analogue; a ecteinascidin analogue. In an embodiment, the mitomycin is selected from: Mitomycin A; Mitomycin C; Porfiromycin; and KW-2149. In an embodiment, the a carmeth izole analogue is selected from: Bis-(Hydroxymethyl)pyrrolizidine; and NSC 602668. In an embodiment, the ecteinascidin analogue is Ecteinascidin 743.

[192] Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.

[193] Cyclopropaindolones and duocamycins may be defined as adenine-specific alkylators. In an embodiment, the cytotoxic or cytostatic agent is a cyclopropaindolone analogue. Preferably, the drug is selected from: adozelesin; and carzelesin.

[194] In an embodiment, the cytotoxic or cytostatic agent is a benz[e]indolone. Preferably, the cytotoxic or cytostatic agent is selected from: CBI-TMI; and iso-CBI.

[195] In an embodiment, the cytotoxic or cytostatic agent is bizelesin. In an embodiment, the cytotoxic or cytostatic agent is a Marine Antitumour Drug. Marine Antitumour Drugs has been a developing field in the antitumour drug development arena (I. Bhatnagaret al, Mar. Drugs 2010, 8, P2702-2720 and T. L. Simmons et al, Mol. Cancer Ther. 2005, 4(2), P333-342). Marine organisms including sponges, sponge-microbe symbiotic association, gorgonian, actinomycetes, and soft coral have been widely explored for potential anticancer agents.

[196] In an embodiment, the cytotoxic or cytostatic agent is selected from: Cytarabine, Ara-C; Trabectedin (ET-743); and EribulinMesylate. In an embodiment, the EribulinMesylate is selected from: (E7389); Soblidotin (TZT 1027); Squalamine lactate; CemadotinPlinabulin (NPI-2358); Plitidepsin; Elisidepsin; Zalypsis; Tasidotin, Synthadotin; (ILX-651); Discodermolide; HT1286; LAF389; Kahalalide F; KRN7000; Bryostatin 1 ; Hemiasterlin (E7974); Marizomib; Salinosporamide A; NPI-0052); LY355703; CRYPTO 52; Depsipeptide (NSC630176); Ecteinascidin 743; Synthadotin; Kahalalide F; Squalamine; Dehydrodidemnin B; Didemnin B; Cemadotin; Soblidotin; E7389; NVP-LAQ824; Discodermolide; HTI-286; LAF-389; KRN-7000 (Agelasphin derivative); Curacin A; DMMC; Salinosporamide A; Laulimalide; Vitilevuamide; Diazonamide; Eleutherobin; Sarcodictyin; Peloruside A; Salicylihalimides A and B; Thiocoraline; Ascididemin; Variolins; Lamellarin D; Dictyodendrins; ES-285 (Spisulosine); and Halichondrin B.

[197] The following cytotoxic or cytostatic agent are also encompassed by the present invention : Amatoxins (a-amanitin)-bicyclic octapeptides produced by basidiomycetes of the genus Amanita, e.g., the Green Deathcap mushroom; Tubulysins; Cytolysins; dolabellanins; Epothilone A, B, C, D, E, F. Epothilones - constitute a class of non-taxane tubulin polymerisation agents and are obtained by natural fermentation of the myxobacteriumSorangiumcellulosum. These moieties possess potent cytotoxic activity which is linked to the stabilisation of microtubules and results in mitotic arrest at the G2/M transition. Epothilones have demonstrated potent cytotoxicity across a panel of cancer cell lines and has often exhibited greater potency than paclitaxel (X. Pivot et al, European Oncology, 2008;4(2), P42-45). In an embodiment, the drug is amatoxin. In an embodiment, the drug is tubulysin. In an embodiment, the drug is cytolysin. In an embodiment, the drug is dolabellanin. In an embodiment, the drug is epothilone.

[198] The following cytotoxic or cytostatic agent are also encompassed by the present invention . In an embodiment, the drug is selected from: Doxorubicin; Epirubicin; Esorubicin; Detorubicin; Morpholino-doxorubicin; Methotrexate; Methopterin; Bleomycin; Dichloromethotrexate; 5- Fluorouracil; Cytosine-p-D-arabinofuranoside; Taxol; Anguidine; Melphalan; Vinblastine; Phomopsin A; Ribosome-inactivating proteins (RIPs); Daunorubicin; Vinca alkaloids; Idarubicin; Melphalan; Cis-platin; Ricin; Saporin; Anthracyclines; Indolino-benzodiazepines; 6- Mercaptopurine; Actinomycin; Leurosine; Leurosideine; Carminomycin; Aminopterin; Tallysomycin; Podophyllotoxin; Etoposide; Hairpin polyamides; Etoposide phosphate; Vinblastine; Vincristine; Vindesine; Taxotere retinoic acid; N8-acetyl spermidine; Camptothecin; Esperamicin; and Ene-diynes.

[199] In one embodiment, the cell killing portion is a peptide toxin, for example an auristatin such as MMAE or MMAF. In one embodiment, the bispecific binding molecule comprises a binding portion and a cell killing portion, wherein the binding portion is a first and second antibody or fragment thereof as described herein and wherein the cell killing portion is a peptide toxin, for example an auristatin such as Auristatin E (AE); Monomethylauristatin E (MMAE); Auristatin F (MMAF), vcMMAE, vcMMAF, mcMMAE and mcMMAF.

[200] Techniques for conjugating cytotoxic or cytostatic agents to proteins, and in particular to antibodies, are well-known. (See, e.g., Alley et ah, Current Opinion in Chemical Biology 2010 14: 1-9; Senter, Cancer J., 2008, 14(3): 154-169.). In certain embodiments, a linking group is used to conjugate the payload, for example a cell killing agent, an immune-modulating payload, a macrophage class switching agent or a light activatable payload, to the first and/or second antibody or antigen-binding fragment, as appropriate. The linker can be cleavable under intracellular conditions, such that cleavage of the linker releases the payload from the binding portion in the intracellular environment. The cleavable linker can be, e.g., a peptidyl linker that is cleaved by an intracellular peptidase or protease enzyme, including a lysosomal or endosomal protease. Cleaving agents can include cathepsins B and D and plasmin (see, e.g., Dubowchik and Walker, Pharm. Therapeutics 83:67-123, 1999). Most typical are peptidyl linkers that are cleavable by enzymes that are present in NTB-A-expressing cells. For example, a peptidyl linker that is cleavable by the thiol-dependent protease cathepsin-B, which is highly expressed in cancerous tissue, can be used (e.g., a linker comprising a Phe-Leu or a Val-Cit peptide).

[201] The cleavable linker can be pH-sensitive, i.e., sensitive to hydrolysis at certain pH values. Typically, the pH- sensitive linker is hydrolysable under acidic conditions. For example, an acid- labile linker that is hydrolysable in the lysosome (e.g., a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide, orthoester, acetal, ketal, or the like) can be used.

[202] Other linkers are cleavable under reducing conditions (e.g., a disulfide linker). The cleavable linker can also be a malonate linker (Johnson et al, Anticancer Res. 15 :1387-93, 1995), a maleimidobenzoyl linker (Lau et al, Bioorg-Med-Chem. 3: 1299-1304, 1995), or a 3' -N-amide analogue (Lau et al, Bioorg-Med-Chem. 3: 1305-12, 1995).

[203] In some embodiments the linker can be a protease cleavable linker, for example a valinecitrulline, which may be cleaved by cathepsin B in the lysosome. [204] The linker also can be a non-cleavable linker, such as a maleimidoca-proyl (me) linker or maleimido-alkylene- or maleimide-aryl linker that is directly attached to the therapeutic agent and released by proteolytic degradation of the binding portion.

[205] The payload may be conjugated to the antigen-binding moiety and/or the C-terminal domain of PCSK9. Preferably, the payload may be conjugated to the antigen-binding moiety.

[206] The payload may be conjugated to the antigen-binding moiety by several routes, employing organic chemistry reactions, conditions, and reagents known to those skilled in the art, including: (1) reaction of a nucleophilic group or an electrophilic group of an antibody or antigen-binding fragment with a bivalent linker reagent, to form antibody-linker intermediate Ab-L, via a covalent bond, followed by reaction with an activated drug moiety D; and (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of an antibody or antigen-binding fragment. Conjugation methods (1) and (2) may be employed with a variety of antibody or antigen-binding fragments, drug moieties, and linkers. Equivalent methods may be employed to conjugate a payload to the C-terminal domain of PCSK9. For example, the method may include (1) reaction of a nucleophilic group or an electrophilic group of the C-terminal domain of PCSK9 with a bivalent linker reagent, to form protein-linker intermediate, via a covalent bond, followed by reaction with an activated drug moiety D; and (2) reaction of a nucleophilic group or an electrophilic group of a drug moiety with a linker reagent, to form drug-linker intermediate D-L, via a covalent bond, followed by reaction with the nucleophilic group or an electrophilic group of the C-terminal domain of PCSK9. Conjugation methods (1) and (2) may be employed with a variety of C-terminal domain of PCSK9 homologs, drug moieties, and linkers.

[207] The linkers may be attached via nucleophilic groups on the antigen-binding moiety or C- terminal domain of PCSK9, as appropriate. Such groups include, but are not limited to: (i) N- terminal amine groups, (ii) side chain amine groups, e.g. lysine, (iii) side chain thiol groups, e.g. cysteine, and (iv) sugar hydroxyl or amino groups where the antigen-binding moiety or C-terminal domain of PCSK9, as appropriate, is glycosylated. Amine, thiol, and hydroxyl groups are nucleophilic and capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups. Certain antibodies have reducible interchain disulfides, i.e., cysteine bridges. Antibodies may be made reactive for conjugation with linker reagents by treatment with a reducing agent.

[208] Additional nucleophilic groups can be introduced into the antigen-binding moiety or C- terminal domain of PCSK9, as appropriate, through the reaction of lysines with 2-iminothiolane (Traut's reagent) resulting in conversion of an amine into a thiol.

[209] Antibody-drug conjugates may also be produced by modification of the antigen-binding moiety or C-terminal domain of PCSK9, as appropriate, to introduce electrophilic moieties, which can react with nucleophilic substituents on the linker reagent or drug. The sugars of glycosylated antibodies may be oxidized, e.g., with periodate oxidizing reagents, to form aldehyde or ketone groups which may react with the amine group of linker reagents or drug moieties. The resulting imine Schiff base groups may form a stable linkage, or may be reduced, e.g., by borohydride reagents to form stable amine linkages. In one embodiment, reaction of the carbohydrate portion of a glycosylated first and/or second antibody or antigen-binding fragment, as appropriate, with either galactose oxidase or sodium meta-periodate may yield carbonyl (aldehyde and ketone) groups in the protein that can react with appropriate groups on the drug. In another embodiment, proteins containing N-terminal serine orthreonine residues can react with sodium meta-periodate, resulting in production of an aldehyde in place of the first amino acid. Such aldehyde can be reacted with a drug moiety or linker nucleophile.

[210] Likewise, nucleophilic groups on a drug moiety include, but are not limited to: amine, thiol, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, and arylhydrazide groups capable of reacting to form covalent bonds with electrophilic groups on linker moieties and linker reagents including: (i) active esters such as NHS esters, HOBt esters, haloformates, and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, carboxyl, and maleimide groups.

[211] Methods for conjugating the payload to the antigen-binding moiety or C-terminal domain of PCSK9, as appropriate, may utilise the presence of one or more non-naturally occurring amino acids. The payload and/or linker may be attached via a non-naturally occurring amino acid residue present in the antigen-binding moiety or C-terminal domain of PCSK9. Methods to introduce non- naturally occurring amino acid residues are known in the art.

[212] The non-naturally occurring amino acid residue to which the payload or linker is attached is may be selected from but not limited to a cysteine residue, a lysine residue, a histidine residue, a tyrosine residue, formylglycine residue.

[213] Where a canonical amino acid is used to attach the payload or linker, site directed mutagenesis may be used to introduce a suitable amino acid residue at a suitable position within the binding molecule. In an embodiment a non-canonical amino acid is used to attach the payload or linker. A “non-canonical” amino acid refers to one of the non-proteinogenic (unnatural) amino acids i.e., an amino acid which is not introduced via the cell’s natural translation machinery. There are many examples of non-canonical amino acids in the art many of which provide a bio- orthogonal handle on which to attach a payload. Where a non-canonical amino acid is used to attach the payload linker, suitable techniques are known in the art to introduce such non-canonical amino acid residues such as chemical modification, tRNA suppressor technology, engineered tRNA,/tRNA synthetase pairs.

[214] The payload may be attached at various positions within the conjugate. One or more copies of the payload may be attached to the conjugate, for example 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 copies of the payload may be attached. Method

[215] According to a second aspect of the present invention there is provided a method of degrading a cell surface receptor that is constitutively internalised and recycled, the method comprising contacting the receptor with a conjugate according to the first aspect of the present invention; wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.

[216] According to a third aspect of the present invention there is provided a method of inhibiting the growth of a cell having a receptor on the surface thereof, the method comprising contacting the cell with a conjugate according to the first aspect of the present invention; wherein the receptor is constitutively internalised and recycled; wherein the antigen-binding moiety is operable to bind to the receptor; and wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.

[217] Suitable features of the second and third aspects of the invention are as provided herein in relation to the first aspect of the invention.

[218] The cell may be a tumour cell. Thus, the conjugate may be operable to bind to a cancer cell. The cancer may be selected from lung cancer, breast cancer, ovarian cancer, bowel cancer, prostate cancer, bladder cancer, colorectal cancer, pancreas carcinoma, kidney cancer, renal cancer, leukaemias, multiple myeloma, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's Lymphoma), brain cancer and other CNS and intracranial tumours cancer, head and neck cancer, oesophageal cancers, solid tumours such as sarcoma and carcinomas, mesothelioma, osteosarcoma, endometrial cancer or melanoma.

[219] In certain embodiments, the cancer may be brain cancer.

[220] In certain embodiments, the tumour cell may be a glioblastoma.

[221] The method may comprise administering a conjugate and/or composition of the invention to a subject.

[222] The term "subject" or "patient" refers to an animal which is the object of diagnosis, treatment, observation, or experiment. By way of example only, a subject includes, but is not limited to, a mammal, including, but not limited to, a human or a non-human mammal, such as a non-human primate, murine, bovine, equine, canine, ovine, or feline. The subject is preferably a human. The subject may be male or female. The subject may be an infant, a toddler, a child, a young adult, an adult or a geriatric. The subject may be a smoker, a former smoker or a non- smoker. The subject may have a personal or family history of cancer. The subject may have a cancer-free personal or family history. The subject may exhibit one or more symptoms of a disease, such as a cancer.

[223] Administration may by any convenient route, including but not limited to oral, topical, parenteral, sublingual, rectal, vaginal, ocular, intranasal, pulmonary, intradermal, intravitrial, intramuscular, intraperitoneal, intravenous, subcutaneous, intracerebral, transdermal, transmucosal, or topical, particularly to the ears, nose, eyes, or skin or by inhalation. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered orally, for example as a liquid, capsule or tablet, such as a slow release formulation. A skilled person would know that the route of administration depends on the disease of interest and the target antigen. For instance, if the target antigen is present in the gastrointestinal tract, oral administration is preferable, while in case of hepatic expression either oral or intravenous administration could constitute viable options. In the case of a lung disease, such as lung cancer oral administration or inhalation may be used.

Use

[224] According to a fourth aspect of the present invention there is provided the use of a conjugate according to the first aspect of the present invention to degrade a cell surface receptor that is constitutively internalised and recycled.

[225] According to a fifth aspect of the present invention there is provided the use of a conjugate according to the first aspect of the present invention to inhibit the growth of a cell having receptor on the surface thereof; wherein the receptor is constitutively internalised and recycled; and wherein the antigen-binding moiety is operable to bind to the receptor.

[226] Suitable features of the fourth and fifth aspects of the invention are as provided herein in relation to the first, second and/or third aspects of the invention.

Further Aspects

[227] According to a sixth aspect of the present invention there is provided a conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof, wherein the C-terminal domain of PCSK9 consists of an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4, or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36, SEQ ID NO. 39.

[228] The antigen-binding moiety may bind to any suitable antigen. The antigen-binding moiety may be capable of binding to one or more antigen(s) expressed by cells, such as by cancer cells, for example. As such, the antigen-binding moiety suitably binds to one or more antigen(s) expressed by cells. Suitable antigens include, but are not limited to, cell surface receptors, such as any of the cell surface receptors defined herein, for example.

[229] The antigen-binding moiety may comprise an antibody. Examples of suitable antibodies are as defined herein in relation to the first, second, third, fourth and/or fifth aspects of the present invention.

[230] The conjugate may comprise a linker. Preferably, the linker may join the antigen-binding moiety and the C-terminal domain of PCSK9. Examples of suitable linkers are as defined herein in relation to the first, second, third, fourth and/or fifth aspects of the present invention.

[231] The conjugate may further comprise a payload. Examples of suitable payloads are as defined herein in relation to the first, second, third, fourth and/or fifth aspects of the present invention. The payload may be attached to the conjugate via a cleavable linker. Examples of suitable cleavable linkers are as defined herein in relation to the first, second, third, fourth and/or fifth aspects of the present invention.

Composition

[232] The invention also includes a composition comprising a conjugate of the invention. The composition may comprise a conjugate of the invention and optionally a pharmaceutically acceptable carrier or excipient.

[233] The composition may comprise one or more conjugate of the invention. The composition may comprise only one conjugate of the invention. The composition may comprise two or more conjugates of the invention.

[234] The pharmaceutically acceptable carrier or vehicle can be particulate, so that the compositions are, for example, in tablet or powder form. The term “carrier” refers to a diluent, adjuvant or excipient, with which conjugate of the present invention is administered. Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. The carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like. In addition, auxiliary, stabilizing, thickening, lubricating and colouring agents can be used. When administered to a human or animal subject, such as a human subject, the conjugates, compositions and/or pharmaceutically acceptable carriers may suitably be sterile. Water is a preferred carrier when the compound of the present invention is to be administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical carriers also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The present compositions, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.

[235] The composition can be in the form of a liquid, e.g., a solution, emulsion or suspension. The liquid can be useful for delivery by injection, infusion (e.g., IV infusion) or sub-cutaneously.

[236] When intended for oral administration, the composition may be in solid or liquid form, where semi-solid, semi-liquid, suspension and gel forms are included within the forms considered herein as either solid or liquid.

[237] As a solid composition for oral administration, the composition can be formulated into a powder, granule, compressed tablet, pill, capsule, chewing gum, wafer or the like form. Such a solid composition typically contains one or more inert diluents. In addition, one or more of the following can be present: binders such as carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, or gelatin; excipients such as starch, lactose or dextrins, disintegrating agents such as alginic acid, sodium alginate, corn starch and the like; lubricants such as magnesium stearate; glidants such as colloidal silicon dioxide; sweetening agents such as sucrose or saccharin; a flavoring agent such as peppermint, methyl salicylate or orange flavoring; and a coloring agent. When the composition is in the form of a capsule (e. g. a gelatin capsule), it can contain, in addition to materials of the above type, a liquid carrier such as polyethylene glycol, cyclodextrin or a fatty oil.

[238] The composition can be in the form of a liquid, e. g. an elixir, syrup, solution, emulsion or suspension. The liquid can be useful for oral administration or for delivery by injection. When intended for oral administration, a composition can comprise one or more of a sweetening agent, preservatives, dye/colorant and flavor enhancer. In a composition for administration by injection, one or more of a surfactant, preservative, wetting agent, dispersing agent, suspending agent, buffer, stabilizer and isotonic agent can also be included.

[239] Compositions can take the form of one or more dosage units.

[240] As used herein, the term “effective amount” means an amount of conjugate that, when administered to a cell, tissue, or subject, is effective to achieve the desired diagnostic and/or therapeutic effect under the conditions of administration. For example, when the conjugate is intended for use as a diagnostic agent, the term “effective amount” is intended to denote a non- lethal but sufficient amount to allow diagnosis of the disorder. When the conjugate is intended for use as a therapeutic agent, the term “effective amount” is intended to denote one which eliminates or diminishes the symptoms associated with the disorder (e.g., by eliminating or reducing the size of a tumour, for example). An effective amount may be determined by one of ordinary skill in the art, using routine experimentation.

[241] In addition, in vitro or in vivo assays can optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the compositions will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease may be taken into account.

Selected definitions

[242] As used herein, unless otherwise expressly specified, all numbers such as those expressing values, ranges, amounts or percentages may be read as if prefaced by the word "about", even if the term does not expressly appear. Also, the recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1 , 2, 3, 4 when referring to, for example, a number of elements, and can also include 1.5, 2, 2.75 and 3.80, when referring to, for example, measurements). The recitation of end points also includes the end point values themselves (e.g. from 1 .O to 5.0 includes both 1.0 and 5.0). Any numerical range recited herein is intended to include all sub-ranges subsumed therein. [243] The terms "comprising", "comprises" and "comprised of’ as used herein are synonymous with "including", "includes" or "containing", "contains", and are inclusive or open-ended and do not exclude additional, non-recited members, elements or method steps.

[244] As used herein, the term "and/or," when used in a list of two or more items, means that any one of the listed items can be employed by itself or any combination of two or more of the listed items can be employed. For example, if a list is described as comprising group A, B, and/or C, the list can comprise A alone; B alone; C alone; A and B in combination; A and C in combination, B and C in combination; or A, B, and C in combination.

EXAMPLES

Selected abbreviations

[245] PCTD: C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9)

Materials and Methods

[246] Vectors: The gene encoding human transferrin receptor tagged at the C-terminus with enhanced green fluorescent protein (EGFP; pLXIN2-TfR-GFP) was amplified from the vector pBA-TfR-GFP (Addgene plasmid #45060; http://n2t.net/addgene:45060; RRID:Addgene_45060; Burack, Silverman, and Banker 2000) using primers that appended in-frame BamHI and Notl restriction sites and subcloned via these sites into a modified pLXIN2 vector (University of Cambridge).

[247] Genes encoding the PCTD-nanobody fusions PCTD-aBC2 and PCTD-aGFP (SEQ ID NOs. 15 and 16, respectively), and the genes encoding the OKT9 Fab fragments VL-CL (SEQ ID NO. 10), VH-CH1-PCTD (“OKT9FabPCTD”; SEQ ID NO. 17) and VH-CH1-2HA (“OKT9Fab2HA”; SEQ ID NO. 18), were obtained as synthetic DNA blocks (gBIocks; Integrated DNA Technologies) with in-frame Agel/Kpnl restriction sites (except for OKT9 VL-CL, which has a stop codon before the Kpnl site) and subcloned into the vector pHLSec (Addgene plasmid #99845; http://n2t.net/addgene:99845; RRID:Addgene_99845; Aricescu, Lu, and Jones 2006) via these sites to yield secreted constructs with C-terminal hexahistidine tags (except for the OKT9 VL-CL fragment; SEQ ID NOs 19, 20 and 21 for VL-CL, OKT9FabPCTD and OKT9Fab2HA as expressed, respectively).

[248] Genes encoding the BC2-CD8a-tagged NPXY, AMOTIF and YQRL reporter constructs were obtained as synthetic DNA blocks (gBIocks; Integrated DNA Technologies; SEQ ID NOs 22, 23 and 24, respectively) and subcloned into modified PB-T PiggyBac cargo vectors (University of Cambridge) such that the resultant reporter constructs were under the control of a constitutive chicken b-actin promoter (with CMV enhancer and chicken p-actin/ra bbit -g lobin chimeric intron) flanked by PiggyBac Transposase recognition sequences. [249] Genes encoding the nanobodies aBC2 and aGFP were obtained as gBIocks (Integrated DNA Technologies; SEQ ID NOs 25 and 26, respectively) and subcloned into an E. coli expression vector, pMW1 .

Creation of cell lines

[250] piggyBac construction of BC2-CD8a reporter cell lines: Cargo vectors encoding the NPXY, AMOTIF and YQRL reporter constructs under the control of a constitutive chicken b-actin promoter (with CMV enhancer and chicken p-actin/rabbit p-globin chimeric intron) flanked by PiggyBac Transposase recognition sequences was mixed with Super PiggyBac transposase vector (System Biosciences) in a 5:1 mass ratio (cargo:transposase) and transfected into HeLa cells (grown in a 6-well dish) using a TransIT-HeLaMONSTER transfection kit according to the manufacturer's protocol. After 48 hours the cells were dissociated with Accutase and plated onto a T75 tissue culture flask. Once near confluence the cells were dissociated with Accutase, pelleted by centrifugation at 300g, the pellet washed with PBS and gently resuspended in PBS supplemented with 1% FBS, and the cells sorted for GFP fluorescence on a Becton Dickinson FACSMelody sorter.

[251] Construction of TfR-GFP reporter cell line by retroviral transduction: Transient DNA transfections were carried out using TransIT-HeLaMONSTER® kit (Mirus, Cambridge Bioscience Ltd, UK) following the manufacturer’s instructions. For stable cell line generation using the pLXlN vectors, HEK 293ET cells were co-transfected with the appropriate retroviral vector and the packaging plasmids pMD.GagPol and pMD.VSVG (University of Cambridge) in the ratio of 50:30:15. Virus-containing supernatant was filtered through a 0.45 pm filter and applied directly to the target cells. The cells were subsequently sorted for GFP fluorescence on a Becton Dickinson FACSMelody sorter.

[252] HEK F expression of proteins by transient transfection: pHLSec vectors containing PCTD-nanobody fusions or OKT9 fab fragments were transfected into 30ml HEK F cells (Thermo Fisher) at a density of 1x10⁶ cells/ml with 30pg DNA using 293Fectin (Thermo Fisher), following the manufacturer's instructions. In the case of the OKT9 fab fragments, 15pg of each vector were mixed and transfected in the same way. After 72 hours supernatants were harvested, passed through a 0.4pm filter, bound to NiNTA agarose (Qiagen), washed with Wash buffer (20mM Tris pH 8, 300mM NaCI), eluted with Wash buffer supplemented with 300mM imidazole pH 8, concentrated and dialyzed against PBS. Aliquots were flash frozen in liquid nitrogen.

[253] E. coli expression of nanobodies: pMW vectors encoding the aBC2 and aGFP nanobodies were expressed in E. coli strain BL21 (DE3)pLysS and purified by NiNTA affinity chromatography as for the HEK F-expressed proteins. [254] Antibodies: The following antibodies were used:

Anti-GFP (Abeam, ab6556, rabbit polyclonal);

Anti-actin (Sigma, A2066, rabbit polyclonal);

Goat Anti-Rabbit IgG H&L (Alexa Fluor® 680) pre-adsorbed (Abeam, ab186696);

APC-labelled mouse monoclonal anti-CD8 a chain, RPA-T8 (eBioscience); and Anti-TfR (Abeam, RabMAb EPR4013).

[255] HEK F expression: HEK F cells were maintained in Freestyle medium. For expression of PCTD fusions, 30ml of cells (3x107 cells) was transfected with 293Fectin according to the manufacturer's protocol. After 3 days, supernatants were collected and filtered through 0.4pm syringe filters. Protease inhibitors (complete EDTA-free inhibitor cocktail, 1 tablet per 30ml expression, plus AEBSF at 25pg/ml) were added and the supernatants incubated with NiNTA (Qiagen) for 1 hour with stirring at 4°C. NiNTA beads were collected in a glass column, washed with 50 ml of 20mM Tris pH 8, 300 mM NaCI, and bound protein eluted with 20mM Tris pH 8, 300 mM NaCI, 300 mM imidazole (pH 8), concentrated in Amicon Ultra-15 concentrators and dialyzed against Dulbecco's PBS.

[256] Western Blot analysis: Adherent cells growing in 6-well dishes were dissociated with Accutase, collected into microcentrifuge tubes and pelleted at 300g for 5 minutes. The supernatants were aspirated and the pellets frozen on dry ice. The pellets were then lysed by resuspension in NP40 lysis buffer (50 mM Hepes pH 7.4, 150 mM NaCI, 1 % NP40) and incubation on ice for 30 minutes with occasional mixing by vortexing. The suspension was then centrifuged at 7000g for 3 minutes at 4°C and the supernatants retained. Total protein concentration was measured using a BCA assay (Pierce) and typically 40pg of protein was loaded on a Bio-Rad TGX 4-20% SDS-PAGE gel and blotted using a Bio-Rad wet transfer system onto Amersham Protran nitrocellulose membranes for 2 hours at 70V constant. The membranes were blocked for 1 hour at 21 °C with PBS + 5% skimmed milk powder (Marvel). Anti-GFP, anti-actin and anti-TfR were used at 1 :1000 dilutions into PBS + 5% skimmed milk powder + 0.1 % Tween-20, and incubated with the blot overnight at 4°C. Blots were washed three times with PBS + 0.1 % Tween- 20 (5 minutes per wash) before addition of goat Anti-Rabbit IgG H&L (Alexa Fluor® 680) diluted 1 :1000 in PBS + 5% skimmed milk powder + 0.1 % Tween-20 and incubation for 1 hour at 21 °C. Blots were washed three times as above and imaged using a Li-Cor Odyssey near-infrared fluorescence imaging system. Blot bands were quantitated by densitometry using Fiji.

[257] General cell treatment procedure: For treatments with PCTD fusion proteins, cells were seeded in 6-well dishes (Falcon) at a density of ~0.3x106 cells/well in DMEM supplemented with 10% Fetal Bovine Serum, Penicillin (100 Units/ml) and Streptomycin (100 pg/ml). Cells were grown for 24 hours, the medium was aspirated and replaced with 1 ml fresh DMEM supplemented as above, and protein treatments added in equal volumes of Dulbecco's PBS; for 'untreated' cell samples, an equal volume of Dulbecco's PBS was added. 5pg/ml concentrations of PCTD- nanobody fusions were used unless otherwise indicated, or a molar equivalent (120 nM) of the nanobody. 'Overnight' treatments were typically done for 16 hours before cells were lysed. Where used, Bafilomycin A1 (or an equal volume of DMSO) was added at 100nM 2 hours prior to treatment with PCTD fusions or control antibodies.

[258] Flow cytometry: Data was collected on a Becton Dickinson LSR Fortessa and analysed with FlowJo software. GFP fluorescence was excited with the 488nm laser and measured with the 530/30nm bandpass filter. APC fluorescence was excited with the 640nm laser and measured with the 670/14 bandpass filter. For the anti-CD8 uptake experiment, compensation was performed using Becton Dickinson CompBeads (following the manufacturer’s instructions) with APC-labelled anti-CD8 alpha chain, and untreated TGN cells (for GFP fluorescence), and applied with the FlowJo software. Where applicable, transduced HeLa cells were sorted on a Becton Dickinson FACSMelody according to the manufacturer's protocols. Briefly, for sorting of stably- transfected GFP-tagged reporter cell lines, nonfluorescent cells were excluded using a gate set to exclude the whole of a control population of unmodified HeLa cells.

[259] Anti-CD8 feeding experiment: Reporter cells, and unmodified HeLa as a negative control, were seeded in 6-well dishes (in 1 ml DMEM + 10% FBS + Penicillin (100 Units/ml) and Streptomycin (100 pg/ml)) at a density of 0.6x106 cells/well and grown overnight (to ~70% confluence). The medium was then supplemented with 5pg APC-labelled mouse monoclonal anti- CD8 alpha chain (RPA-T8; commercially available from eBioscience) and the cells incubated for a further 30 minutes at 37°C/5% CO2. Cells were then placed on ice, washed twice with cold PBS, then dissociated with trypsin. The cells were pelleted by centrifugation and washed once with PBS, and then analysed by flow cytometry.

[260] Cell culture and virus infection: All cells were incubated at 37°C in 5% CO2. Human fetal foreskin fibroblasts (Hfff2, ECACC 86031405) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Merck) supplemented with sterile-filtered 10% heat-inactivated foetal bovine serum (FBS; PAN Biotech) and penicillin (100U/ml)/streptomycin (100ug/ml) (Merck). Kasumi-3 cells (ATCC, CRL-2725) were grown in Roswell Park Memorial Institute (RPMI) medium-1640 (Merck), with sterile-filtered 20% heat-inactivated FBS (PAN Biotech) and penicillin (100U/ml)/streptomycin (100ug/ml) (Merck). Kasumi-3 cells were infected with viral isolate RV 1664 (HCMV-TB40/E-IE2-eYFP)2 or viral isolate HCMV-TB40/E-US28-GFP at a predicted multiplicity of infection (MOI) of 3. Hfff2 were infected with viral isolate HCMV-TB40/E-US28-GFP at an MOI of 0.1-1.

[261] Differentiation of cells: Cell differentiation with phorbol 12-myristate 13-acetate (PMA, Merck) was achieved at a concentration of 20 ng/mL. [262] Fluorescence microscopy assays: Fibroblasts were infected with HCMV-TB40/E-US28- GFP and were left untreated or were treated with PCTD-Vun100bv (500 nM; SEQ ID NO.s 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed) or a non-US28- targeting PCTD-aBC2 control (500 nM; SEQ ID NO. 15) three days post-infection. To assess US28 degradation over a time course of 29h, pictures were taken every 30 minutes using live-cell microscopy (Cellomics ArrayScan XTI, ThermoFisher), starting 30 minutes after treatment. The color of the pictures (S2a) was inverted to highlight the decrease in GFP intensity over time. The decay in fluorescent intensity signal for each spot was normalised to the mean intensity of each spot measured at the first time point.

[263] Kasumi-3 cells were infected with HCMV-TB40/E-US28-GFP. Two days post-infection, cells were treated with PCTD-Vun100bv (500 nM; SEQ ID NO.s 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed), or were left untreated. Pictures were taken three days post-treatment. Brightness measurements were performed using Imaged (version 1.53e). The mean spot intensity of infected cells was measured by selecting representative cells with the freehand selection tool of Imaged, then subtracting the mean background intensity for each field of view. The measured reporter fluorescence of treated cells was normalized to the average fluorescence of untreated cells.

[264] US28 degradation assay via flow cytometry: US28 expression was assessed by measuring the GFP intensity of PCTD-Vun100bv-treated fibroblasts by flow cytometry. To do this, fibroblasts were infected with HCMV-TB40/E-US28-GFP. Three days post-infection, cells were treated with PCTD-Vun100bv (500 nM; SEQ ID NOs 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed), Vun100bv (500 nM; SEQ ID NO. 7), or were left untreated. Two days post-treatment, adherent fibroblasts were washed with PBS, dissociated with Trypsin- EDTA, and transferred to FACS tubes. Cells were analyzed for GFP with a BD Accuri C6 Plus flow cytometer and BD Accuri C6 software (version 1 .0.264.21).

[265] Detection of IE expression: Kasumi-3 cells were seeded in 96-well plates. The following day, the medium was removed and cells were left uninfected or infected with HCMV-RV1164-IE2- eYFP. Two hours post-infection, the medium was replaced with medium either containing PCTD- Vun1 OObv (500 nM; SEQ ID NOs 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed), PMA at a final concentration of 20 ng/ml (positive control), Vun100bv (100 nM, as described previously; SEQ ID NO.7) or untreated medium (negative control). Three to four days post-infection, IE expression was detected by manually counting for eYFP-expressing cells with a widefield Nikon TE200 microscope. The supernatant was transferred onto fibroblasts three to four days post-treatment, and IE2-eYFP-positive cells were counted four days after supernatant transfer. [266] Statistical analysis: Statistical analysis was performed using GraphPad Prism v9.5.0. Data are plotted as mean ± SD.

Results

Lysosomal degradation

[267] We hypothesized that the ~25 kDa PCTD might be sufficient to drive lysosomal degradation of a target when fused to an antibody that mediates target recognition.

[268] To test this hypothesis, we created a HeLa cell line stably expressing a reporter construct composed of transferrin receptor 1 (TfR) tagged on the extracellular face with GFP, and expressed in HEK-293F cells a chimeric protein composed of an anti-GFP nanobody (aGFP) fused to PCTD (PCTD-aGFP; SEQ ID NO. 27). As a control, aGFP from E. coll was expressed and purified. The TfR-GFP reporter cell line was treated with PCTD-aGFP, aGFP, or left untreated. After overnight incubation, the cells were lysed and cell extracts probed by immunoblot for the presence of TfR-GFP. PCTD-aGFP drove a large (~4.6-fold; ~80%) and significant reduction of TfR-GFP, while no significant degradation was stimulated by aGFP alone (Figures 1a and 1 b).

[269] To demonstrate targeted degradation of an endogenous transmembrane protein, the anti- TfR antibody OKT9 was fused to PCTD. As described in detail above, this was achieved by expressing and purifying a Fab fragment of OKT9 with PCTD fused to the C terminus of the truncated heavy chain (OKT9FabPCTD) via co-transfection of HEK-293F cells with vectors encoding the light chain (VL-CL) and the heavy chain-PCTD fusion (VH-CH1-PCTD). As a control, a modified version of this construct with the PCTD domain replaced by a dual HA (hemagglutinin) tag (OKT9FabHA), was expressed and purified. HeLa cells were treated overnight with OKT9FabPCTD, OKT9FabHA, or left untreated before lysis and analysis by immunoblot (Figures 1c and 1d). OKT9FabPCTD drove a statistically significant and substantial reduction (3.3-fold; 69%) of TfR compared to untreated cells. OKT9FabHA caused a modest decrease (1 .2-fold, 15%) that was not significantly different from untreated cells. These results confirm that PCTD can drive the degradation of proteins expressed at endogenous levels, and that degradation is effective when PCTD is conjugated to either nanobodies or antibody Fab fragments.

Internalisation and Recycling

[270] To probe whether PCTD-driven degradation requires target internalisation and recycling, cell lines were generated expressing a chimeric reporter comprising an extracellular CD8 a-chain tagged with the BC2 peptide (Braun, M. B. et al. Peptides in headlock-a novel high-affinity and versatile peptide-binding nanobody for proteomics and microscopy. Sci. Rep. 6, 19211 (2016)), a single transmembrane helix, a variable cytoplasmic domain, and an intracellular EGFP for fluorescence detection (Figure 2; SEQ ID NO.s. 22, 23 and 24, respectively). This design allowed all cell lines to be treated with the same reagents, eliminating a potential source of variation. PCTD fused to an anti-BC2 nanobody (PCTD-aBC2; SEQ ID NO. 28) was expressed in HEK-293F cells and aBC2 was purified from E. coll. The variable cytoplasmic domains were as follows:

• NPXY: an NPXY-type motif that drives both internalisation by clathrin-mediated endocytosis and PM recycling (Chen, W. J., Goldstein, J. L. & Brown, M. S. NPXY, a sequence often found in cytoplasmic tails, is required for coated pit-mediated internalization of the low density lipoprotein receptor. J. Biol. Chem. 265, 3116-3123 (1990) and Steinberg, F., Heesom, K. J., Bass, M. D. & Cullen, P. J. SNX17 protects integrins from degradation by sorting between lysosomal and recycling pathways. J. Cell Biol. 197, 219-230 (2012));

• AMOTIF: lacks known internalisation motifs;

• YQRL (or TGN): a motif known to direct efficient internalisation from the PM and rapid rerouting from early endosomal compartments to the trans-Golgi network (Bos, K., Wraight, C. & Stanley, K. K. TGN38 is maintained in the trans-Golgi network by a tyrosine-containing motif in the cytoplasmic domain. EMBO J. 12, 2219-2228 (1993)).

[271] All three constructs are accessible by extracellular ligands, confirmed by uptake of fluorescent anti-CD8 antibody (Figure 3). Cell lines were treated overnight with PCTD-aBC2, aBC2 alone, or left untreated, and cellular GFP fluorescence was analysed by flow cytometry to measure degradation of the reporter relative to untreated cells (Figure 4a). Treatment with aBC2 alone did not cause significant degradation of any of the reporters (Figure 4b). Treatment with PCTD-aBC2 provoked significant degradation of the NPXY reporter (1 .5-fold, 34%), but not of the AMOTIF reporter (Figure 4b). The YQRL was also degraded by PCTD-aBC2 (1.1-fold, 9%). These data suggest that PCTD degrades targets that recycle through 'later' endosomal compartments more effectively than those that traffic through the plasma membrane before retrieval to the trans-Golgi network.

[272] To assess whether PCTD drives lysosomal destruction of targets, degradation experiments were performed in the presence or absence of bafilomycin A1 , a compound that inhibits protein hydrolysis by blocking lysosomal acidification. After pre-incubation of NPXY reporter cells with bafilomycin A1 or carrier (DMSO), the cells were treated overnight by supplementing the medium with PCTD-aBC2 or left untreated. PCTD-aBC2 was unable to drive degradation of reporter in the presence of bafilomycin A1 (Figures 5a and 5b), confirming that PCTD stimulates lysosomal degradation of targets.

Human cytomegalovirus (HCMV) protein US28

[273] Next, the technology was applied to a medically relevant target, the human cytomegalovirus (HCMV) protein US28. While primary infection with HCMV is often subclinical, it can cause severe end-organ disease in immunocompromised individuals (Griffiths, P. & Reeves, M. Pathogenesis of human cytomegalovirus in the immunocompromised host. Nat. Rev. Microbiol. 19, 759-773 (2021)). A hallmark of HCMV infection is the establishment of a lifelong latent infection in undifferentiated cells of the myeloid lineage (Sinclair, J. & Sissons, P. Latency and reactivation of human cytomegalovirus. J. Gen. Virol. 87, 1763-1779 (2006) and Poole, E. & Sinclair, J. Sleepless latency of human cytomegalovirus. Med. Microbiol. Immunol. (Bed.) 204, 421-429 (2015)), which cannot be targeted with current HCMV antivirals (Krishna, B. A., Wills, M. R. & Sinclair, J. H. Advances in the treatment of cytomegalovirus. Br. Med. Bull. 131 , 5-17 (2019) and Perera, M. R., Wills, M. R. & Sinclair, J. H. HCMV Antivirals and Strategies to Target the Latent Reservoir. Viruses 13, 817 (2021)). Latency is characterized by the suppression of immediate early (IE) gene expression and the absence of infectious virion production. Reactivation from latency, which depends on the activation of viral IE gene expression, results in the production of infectious particles. The initial switch from latency to lytic infection is controlled by a single viral promoter, the major immediate early promoter (MIEP), which itself is controlled by epigenetic repression (Elder, E. & Sinclair, J. HCMV latency: what regulates the regulators? Med. Microbiol. Immunol. (Bed.) 208, 431-438 (2019)). US28, a viral chemokine receptor expressed during latent and lytic infection, is crucial for mediating the repression of the MIEP during latency.

[274] A virus-specific nanobody (Vun100bv), that functions as a partial inverse agonist of US28 and induces the reactivation of HCMV from latency, has recently been developed (De Groof, T. W. M. et al. Targeting the latent human cytomegalovirus reservoir for T-cell-mediated killing with virus-specific nanobodies. Nat. Common. 12, 4436 (2021)). The induction of viral IE gene expression offers therapeutic potential, as reactivation allows infected cells to be recognized by HCMV-specific cytotoxic T-cells.

[275] US28 undergoes rapid constitutive endocytosis and recycling back to the PM, suggesting that it might be a suitable target for PCTD-driven degradation.

[276] Vun100bv (SEQ ID NO. 7) was fused to the C terminus of PCTD to generate a virus-specific nanobody conjugate (PCTD-Vun100bv; SEQ ID NOs 34 and 35, wherein SEQ ID NO. 35 is the amino acid sequence as expressed). First, we assessed the ability of PCTD-Vun100bv to target and degrade US28 in fibroblasts, a cell type that permits HCMV lytic infection. Fibroblasts were infected with HCMV encoding GFP-tagged US28 (US28-GFP-HCMV) and subsequently treated with PCTD-Vun100bv, the non-US28-targeting PCTD-aBC2 nanobody, or left untreated. US28 expression levels were monitored using live-cell fluorescence microscopy. As early as 60 minutes after treatment with PCTD-Vun100bv, a decrease in the level of US28-specific fluorescence can be observed compared with untreated or PCTD-aBC2 treated cells (Figure 6). To confirm that PCTD was responsible for this effect, we directly compared the effect of PCTD-Vun100bv and Vun100bv on the level of US28 expression. Fibroblasts were infected with US28-GFP-HCMV and then treated with PCTD-Vun100bv, Vun100bv, or left untreated. We observed a distinct loss of total GFP levels in PCTD-Vun100bv treated cells, whereas treatment with Vun100bv had little effect on US28, suggesting that the PCTD modification is responsible for receptor degradation (Figure 7). Since fibroblasts support only lytic infection, we next studied PCTD-Vun100bv treatment in cell types supporting HCMV latency. We repeated our analyses in the Kasumi-3 cell line, a CD34+ myeloblastic cell line known to support HCMV latency and reactivation. In Kasumi- 3 cells, PCTD-Vun100bv treatment promoted degradation of US28, indicated by the reduction of fluorescence intensity (Figure 8).

[277] US28 signalling is essential to repress the HCMV MIEP to establish and maintain latency in Kasumi-3 cells (Humby, M. S. & O’Connor, C. M. Human Cytomegalovirus US28 Is Important for Latent Infection of Hematopoietic Progenitor Cells. J. Virol. 90, 2959-2970 (2015)). To determine if US28 degradation by PCTD-Vun100bv prevents the establishment of latency, we infected Kasumi-3 cells with an HCMV-IE2-eYFP virus that allows the detection of cells expressing lytic IE gene products, and treated cells with nanobodies two hours post-infection. As a positive control, we treated Kasumi-3 cells with phorbol myristate acetate (PMA), which makes them fully permissive for lytic HCMV infection29. PCTD-Vun100bv induced IE expression levels ~3-times higher than the untreated control cells (Figure 9a) and ~1.5-fold higher than the unmodified Vun Obv nanobody. Finally, we assessed if PCTD-Vun100bv drives full lytic infection in Kasumi-3 cells, which might pose a severe risk for immunosuppressed patients. We transferred the supernatant of infected and treated Kasumi-3 cells onto fibroblasts and counted IE expression four days later. Apart from the positive control PMA, we did not see a significant increase in viral production in any of the conditions, confirming an absence of lytic virus replication (Figure 9b).

[278] In summary, we have shown in a variety of cell types and against a range of targets that the PCTD domain is able to direct internalising receptors to the lysosomal degradation pathway. PCTD is a comparatively small domain (~25 kDa) that is independently folded and can be genetically fused to antibody domains for simple recombinant expression. As an endogenous human-secreted protein, PCTD is less likely to elicit a strong immune response. Given the large range of medically-relevant membrane protein targets that undergo recycling to the cell surface, we envisage PCTD as a useful alternative to other targeted membrane protein degradation technologies.

Mutant PCTD (HQ^RR)

[279] A HeLa cell line stably expressing the NPXY reporter construct (see above) was treated overnight with 5pg/ml PCTD-aBC2 (SEQ ID NO. 28) or PCTD(H553R and Q554R)-aBC2 (SEQ ID NO. 29) or left untreated. Cells were then analysed by flow cytometry to assess target degradation by reduction in reporter fluorescence. As shown in Figure 10, there is only a minor difference in degradation (~68% vs 62% fluorescence remaining) between the wild-type and the mutant, although it is significant at the p=0.05 level.

Trastuzumab-PCTD

[280] Two trastuzumab fusion constructs were tested. In one, PCTD was fused to the C-terminus of the light chain (SEQ ID NO. 30) and co-expressed with the heavy chain (SEQ ID 10); in the other, PCTD was fused to the C-terminus of the heavy chain (SEQ ID NO. 31) and co-expressed with the light chain (SEQ ID 11). The PCTD fused to the C-terminus of the heavy chain as expressed in HeLa cells is provided in SEQ ID NO. 32.

SUBSTITUTE SHEET (RULE 26) [281] SKBR3 cells were seeded at a density of 1x10⁶ cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium containing trastuzumab at 5pg/ml, a molar equivalent of trastuzumab-PCTD fusion, or PBS. The following morning the cells were washed in PBS, lysed and 40pg protein extract (quantitated by BCA assay) run on SDS-PAGE gels, blotted onto nitrocellulose, and Erbb2 levels quantitated by Western Blot with Licor fluorescence detection using antibody 29D8 (Cell Signalling, anti-Erbb2, 1 :1000) and goat anti-rabbit Alexa Fluor 680 (Abeam), normalised with anti-GAPDH antibody (GA1 R, Antibodies.com, 1 :5000) and goat anti-mouse Alexa Fluor 780. Bands were quantitated by densitometry using Fiji. Means and standard deviation from 2 experiments shown. The results are shown in Figure 1 1 .

PCTD subdomain experiments

[282] PCTD comprises subdomains defined here as M1 (residues 450-529), M2 (residues 530- 605) and M3 (residues 606-692). A fusion of the M1 subdomain with the aBC2 nanobody (M1- aBC2) was expressed in HEK F cells (SEQ ID NO.33), and tested its ability to cause degradation of a BC2-tagged NPXY reporter construct as follows.

[283] HeLa cells stably expressing a chimeric NPXY reporter, comprising an extracellular CD8 a-chain tagged with the p-catenin-derived BC2 peptide tag, a single transmembrane helix, a cytoplasmic domain comprising an 'NPXY' sorting motif embedded within an unstructured sequence and an intracellular EGFP for fluorescence detection, were seeded at a density of 1x10⁶ cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1-aBC2 or PCTD-aBC2 at 5pg/ml. As a control, cells incubated in untreated (i.e., supplemented) complete medium. The following morning, the cells were washed in PBS, dissociated with Accutase, washed in complete medium, resuspended in PBS, and analysed by flow cytometry. EGFP fluorescence was measured on a Becton Dickinson LSR Fortessa and the data analysed with FlowJo software. The results are shown in Figure 12. NPXY reporter remaining after treatment (measured by EGFP fluorescence) is shown as a % of untreated. Means and standard deviation from 2 experiments shown.

[284] HeLa cells stably expressing a chimeric NPXY reporter, comprising an extracellular CD8 a-chain tagged with the p-catenin-derived BC2 peptide tag, a single transmembrane helix, a cytoplasmic domain comprising an 'NPXY' sorting motif embedded within an unstructured sequence and an intracellular EGFP for fluorescence detection, were seeded at a density of 1x10⁶ cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1-aBC2 or PCTD-aBC2 at 5pg/ml. As a control, cells incubated in untreated (i.e., supplemented) complete medium. The following morning, the cells were washed in PBS, dissociated with Accutase, washed in complete medium, resuspended in PBS, and analysed by flow cytometry. EGFP fluorescence was measured on a Becton Dickinson LSR Fortessa and the data analysed with FlowJo software. The results are shown in Figure 12. NPXY reporter remaining after treatment (measured by EGFP fluorescence) is shown as a % of untreated. Means and standard deviation from 2 experiments shown.

[285] When expressed in isolation, the M1 subdomain displays limited solubility, beginning to precipitate above concentrations of ~1 mg/ml in PBS. To improve solubility of M1 , Trp453, Leu455 and Leu529, which were identified as key hydrophobic residues by removal of M2/M3, were each mutated to serine (M1 SOL1 ; SEQ ID NO. 36). A fusion of M1 SOL1 with the ocBC2 nanobody was expressed in HEK F cells (M1 SOL1-ocBC2; SEQ ID NO. 37, wherein SEQ ID NO. 38 is the amino acid sequence as expressed). This construct displayed improved solubility, not beginning to precipitate until concentrations above ~4mg/ml in PBS. The ability of M1 SOL1-aBC2 to cause degradation of the BC2-tagged NPxY reporter construct was tested as follows.

[286] HeLa cells stably expressing the chimeric NPxY reporter were seeded at a density of 1x10⁶ cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1 SOL1-aBC2 or PCTD-aBC2 at 5pg/ml, or left untreated. The following morning the cells were washed in PBS, dissociated with Accutase, washed in complete medium, resuspended in PBS, and analysed by flow cytometry. EGFP fluorescence was measured on a Becton Dickinson LSR Fortessa and the data analysed with FlowJo software. The results are shown in Figure 13. NPXY reporter remaining after treatment (measured by EGFP fluorescence) is shown as a % of untreated. Means and standard deviation from 2 experiments is shown. One-way ANOVA analysis of mean fluorescence (with Tukey’s method for multiple comparisons) indicates that M1 SOL drives substantial degradation, indicated by significant (* denotes p<0.01) reduction of mean reporter fluorescence, similar to PCTD (ns indicates no significant difference between PCTD and M1 SOL).

[287] Degradation mediated by mutants of PCTD lacking M3 (M1-M2; SEQ ID NO. 4) or M2 (MIMS; SEQ ID NO. 39) was investigated using fusions of these paired subdomains with the aBC2 nanobody (M1-M2-ocBC2 SEQ ID NO. 40, wherein SEQ ID NO. 41 is the amino acid sequence as expressed, and M1-M3-ocBC2 SEQ ID NO. 42, wherein SEQ ID NO. 43 is the amino acid sequence as expressed, respectively).

[288] HeLa cells stably expressing the chimeric NPXY reporterwere seeded at a density of 1x10⁶ cells/well in 6-well dishes. The following day, the cells were washed with PBS and then incubated overnight in complete medium supplemented with M1-M2-aBC2, M1-M3-aBC2 or PCTD-aBC2, at 5pg/ml, or left untreated. The following morning the cells were washed in PBS, dissociated with Accutase, washed in complete medium, resuspended in PBS, and analysed by flow cytometry. EGFP fluorescence was measured on a Becton Dickinson LSR Fortessa and the data analysed with FlowJo software. The results are shown in Figure 14. NPXY reporter remaining after treatment (measured by EGFP fluorescence) is shown as a % of untreated. Means and standard deviation from 2 or 3 (for M1-M2-ocBC2 and M1-M3-ocBC2) experiments shown. One-way ANOVA analysis of mean fluorescence (with Tukey’s method for multiple comparisons) indicates that both M1-M2 and M1-M3 drove degradation, indicated by significant (*** denotes p<0.0001) reduction of mean reporter fluorescence, with the M1-M3 fusion being the most effective.

Degradation of US28 using PCTD-Vun100bv

[289] Kasumi-3 cells were infected with HCMV-TB40/E-US28-GFP virus for 3 days, leading to expression of GFP-tagged US28. Cells were then treated with Vun Obv (500nM) or PCTD- Vun Obv (500 nM) for 2 h and harvested for Western blotting with anti-actin (anti-actin goat primary AbCam, ab8229 1 in 2000) and anti-GFP (anti-GFP rabbit primary AbCam, ab6556 1 in 1000), followed by HRP anti-goat or anti-rabbit secondary antibody (AbCam 1 in 2500). Blots were developed with the use of enhanced chemiluminescence (GE Healthcare) and visualized with autoradiography film. Bands from Western blots were then analysed by densitometry and plotted relative to the actin control and untreated cells, where the graph represents individual and mean values from triplicate samples with standard deviation error bars. Statistical significance was determined using an unpaired t-test, where p < 0.01 = **. p < 0.001 = ***. The results are shown in Figure 15. The results show that PCTD-Vun100bv causes degradation of US28 in a CD34+ cell line.

Claims

1 . A conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9) or a homolog thereof; wherein the antigen-binding moiety is operable to bind to a receptor on the surface of a cell; and wherein the receptor is constitutively internalised and recycled.

2. The conjugate according to claim 1 , wherein the receptor comprises a transmembrane domain.

3. The conjugate according to claim 1 , wherein the C-terminal domain of PCSK9 comprises an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4, or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO. 39; and/or wherein the C-terminal domain of PCSK9 consists of an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4, or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO. 39.

4. The conjugate according to any one of claims 1-3, wherein the antigen-binding moiety is operable to bind to a receptor having one or more internalisation and/or recycling motifs selected from the group consisting of: NXXY, such as NPXY; X^JXXX², YXXX², WXXX², X³XXXLX⁴, KXXM, X⁵XX⁶, wherein X represents any amino acid residue; X¹ represents Y or W; X² represents L, I, V, M or F; X³ represents D, E, Q, N, K, R, or H, such as D or E; and X⁴ represents L, I, V or M, such as L or I; X⁵ represents F, W, Y or H; and X⁶ represents L or M.

5. The conjugate according to claim 4, wherein the internalisation and/or recycling motif is other than KXXM and is located in an unstructured region of the receptor; or wherein the internalisation and/or recycling motif is KXXM and is located in an accessible helix of the receptor, wherein X represents any amino acid residue.

6. The conjugate according to any one of claims 4 or 5, wherein the internalisation and/or recycling motif is located in the cytosolic portion of the receptor.

7. The conjugate according to claim 6, wherein the internalisation and/or recycling motif is located at least 5 residues, such as at least 6 residues, away from the predicted start of the cytosolic portion.

8. The conjugate according to any one of claims 4-7, wherein the internalisation and/or recycling motif is not YQRL.

9. The conjugate according to any one of claims 1-8, wherein the antigen-binding moiety is operable to bind to one or more receptor(s) selected from the group consisting of: HCMV; US28; Erbb2; CAIX; p-glycoprotein; ADAMs, such as ADAM17; integrins; CDs, such as CD22; CD33, CD56, CD70 and CD71 ; GLUT1 ; GPCRs; LDLR; PD-1 ; PD-L1 ; voltage-gated sodium channels, such as Nav1.1 , Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8 and Na_v1.9; TRKA/NTRK1 channels; R-SNAREs, such as VAMP2, VAMP3, VAMP4, VAMP7 and VAMP8; LAMP1 ; LAMP2; HER2; HER3; CTLA-4, Frizzled receptors, such as Frizzled-1 , Frizzled-2, Frizzled-3, Frizzled-4, Frizzled-5, Frizzled-6, Frizzled-7, Frizzled-8, Frizzled-9 and Frizzled-10; IL1 RAP; IL2RA; IL2RB; BACE1 ; BACE2; amyloid beta precursor protein; APLP1 ; APLP2; subunits of the gamma secretase complex, such as APH1 A, APH1 B, Nicastrin, Presenilin and PENSEN; TGFBR1 ; EPHB2; OSMR; MUC1 ; NRP1 ; ROR1 ; ICAM1 ; EPHA3; SEMA4B; GPR107; and VEGFR2.

10. The conjugate according to any one of claims 1-9, wherein the antigen-binding moiety comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11 .

11. The conjugate according to any one of claims 1-10, wherein the antigen-binding moiety is operable to bind to US28.

12. The conjugate according to any one of claims 1-11 , wherein the conjugate comprises a linker between the antigen-binding moiety and the C-terminal domain of PCSK9, for example a GS-linker, for example a linker comprising the sequence (GGGS)_n, wherein n is from 1 to 20, such as from 1 to 10.

13. The conjugate according to any one of claims 1-12 further comprising a payload, such as a cell killing agent, a macrophage class switching agent, an immune-modulating payload a light activatable payload and/or a molecular label.

14. The conjugate according to claim 13, wherein the payload is attached to the conjugate via a cleavable linker.

15. A method of degrading a cell surface receptor that is constitutively internalised and recycled, the method comprising contacting the receptor with a conjugate according to any one of claims 1-14; wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.

16. A method of inhibiting the growth of a cell having receptor on the surface thereof, the method comprising contacting the cell with a conjugate according to any one of claims 1- 14; wherein the receptor is constitutively internalised and recycled; wherein the antigen-binding moiety is operable to bind to the receptor; and wherein the receptor and conjugate are subsequently transported to a lysosome where the receptor is degraded.

17. The method according to any one of claims 15 or 16, wherein the conjugate further comprises a payload, such as a cell killing agent, a macrophage class switching agent, an immune-modulating payload a light activatable payload and/or a molecular label.

18. The method according to claim 17, wherein the payload is attached to the conjugate via a cleavable linker.

19. The method according to any one of claims 15-18, wherein the cell is a tumour cell.

20. The method according to any one of claims 15-19, wherein the receptor comprises a transmembrane domain.

21 . The method according any one of claims 15-20, wherein the C-terminal domain of PCSK9 comprises an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4, or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO.39; and/or wherein the C-terminal domain of PCSK9 consists of an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4, or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO. 39.

22. The method according to any one of claims 15-21 , wherein the antigen-binding moiety is operable to bind to a receptor having one or more internalisation and/or recycling motifs selected from the group consisting of: NXXY, such as NPXY; X¹XXX², YXXX², WXXX², X³XXXLX⁴, KXXM, X⁵XX⁶, wherein X represents any amino acid residue; X¹ represents Y or W; X² represents L, I, V, M or F; X³ represents D, E, Q, N, K, R, or H, such as D or E; and X⁴ represents L, I, V or M, such as L or I; X⁵ represents F, W, Y or H; and X⁶ represents L or M.

23. The method according to claim 22, wherein the internalisation and/or recycling motif is other than KXXM and is located in an unstructured region of the receptor; or wherein the internalisation and/or recycling motif is KXXM and is located in an accessible helix of the receptor, wherein X represents any amino acid residue.

24. The method according to any one of claims 22 or 23, wherein the internalisation and/or recycling motif is located in the cytosolic portion of the receptor.

25. The method according to claim 24, wherein the internalisation and/or recycling motif is located at least 5 residues, such as at least 6 residues, away from the predicted start of the cytosolic portion.

26. The method according to any one of claims 22-25, wherein the internalisation and/or recycling motif is not YQRL.

27. The method according to any one of claims 15-26, wherein the antigen-binding moiety is operable to bind to one or more receptor(s) selected from the group consisting of: HCMV US28; Erbb2; CAIX; p-glycoprotein; ADAMs, such as ADAM17; integrins; CDs, such as CD22; CD33, CD56, CD70 and CD71 ; GLUT1 ; GPCRs; LDLR; PD-1 ; PD-L1 ; voltage-gated sodium channels, such as Nav1.1 , Nav1.2, Nav1.3, Nav1.4, Nav1.5, Nav1.6, Nav1.7, Nav1.8 and Na_v1.9; TRKA/NTRK1 channels; R-SNAREs, such as VAMP2, VAMP3, VAMP4, VAMP7 and VAMP8; LAMP1 ; LAMP2; HER2; HER3; CTLA-4, Frizzled receptors, such as Frizzled-1 , Frizzled-2, Frizzled-3, Frizzled-4, Frizzled-5, Frizzled-6, Frizzled-7, Frizzled-8, Frizzled-9 and Frizzled-10; IL1 RAP; IL2RA; IL2RB; BACE1 ; BACE2; amyloid beta precursor protein; APLP1 ; APLP2; subunits of the gamma secretase complex, such as APH1 A, APH1 B, Nicastrin, Presenilin and PENSEN; TGFBR1 ; EPHB2; OSMR; MUC1 ; NRP1 ; ROR1 ; ICAM1 ; EPHA3; SEMA4B; GPR107; and VEGFR2.

28. The method according to any one of claims 15-27, wherein the antigen-binding fragment comprises an amino acid sequence selected from the group consisting of: SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10 and SEQ ID NO. 11 .

29. The method according to any one of claims 15-28, wherein the antigen-binding moiety is operable to bind to US28.

30. The method according to any one of claims 15-29, wherein the conjugate comprises a linker between the antigen-binding moiety and the C-terminal domain of PCSK9, for example a GS-linker, for example a linker comprising the sequence (GGGS)_n, wherein n is from 1 to 20, such as from 1 to 10.

31 . Use of a conjugate according to any one of claims 1-14 in a method according to any one of claims 15-30.

32. A conjugate comprising an antigen-binding moiety and the C-terminal domain of proprotein convertase subtilisin/kexin type 9 (PCSK9), wherein the C-terminal domain of PCSK9 consists of an amino acid sequence as set forth in SEQ ID NO. 1 , SEQ ID NO. 2, SEQ ID NO. 3 or SEQ ID NO. 4 or a homolog thereof, such as that according to SEQ ID NO. 5, SEQ ID NO. 36 or SEQ ID NO. 39.