Activatable binding molecule comprising an antibody or antigen-binding fragment thereof

Abstract

The invention relates to a system which can be used to inactivate an antibody, fragment thereof or an immunoconjugate, thereby preventing engagement with healthy cells that express the target antigen. Upon entry to the tumour microenvironment the antibody or immunoconjugate becomes activated allowing interaction with the target antigen. The invention relates to an activatable binding molecule, comprising the structure of formula (I): MM-CM-EX-LM-AB (I) wherein AB represents an antibody or antigen binding fragment thereof capable of binding a target when the binding molecule is in an activated state, MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target, CM represents a cleavable moiety, EX represents an extension moiety, and LM represents a linking moiety, wherein the linking moiety is covalently attached via one or more amino acid side chain present in the CHI domain, CL domain, hinge domain and / or Fc domain of AB.

Description

[0001] Activatable Binding Molecule

[0002] Field of Invention

[0003] The present invention relates to activatable binding molecules and associated methods.

[0004] Introduction

[0005] ADCs consist of an antibody guidance system linked to a toxic payload by a linker and this simple design has come to the fore amongst the recent wave of immunotherapeutics as the most capable strategy for effectively treating solid tumour cancers. However, because healthy cells most often express epitope targets of the ADC antibody moiety, albeit at lower levels, they remain as targets of opportunity and are the primary cause of adverse effects associated with ADC treatment. As such there is a need to develop methods to furthertarget ADCs to the tumour and avoid off-target toxicities.

[0006] Summary of the Invention

[0007] The present invention relates to a system which can be used to inactivate an antibody, fragment thereof or an immunoconjugate, thereby preventing engagement with healthy cells that express the target antigen. Upon entry to the tumour microenvironment the antibody or immunoconjugate becomes activated allowing interaction with the target antigen. As such the present invention provides high tumour specificity, obviating collateral adverse effects of antibody or immunoconjugate treatment on healthy cells expressing the target antigen.

[0008] Herein the inventors have developed a “shield system” which can be applied to any antibody or fragment to produce an activatable binding molecule. The shield comprises: a masking moiety (MM) capable of blocking the interaction with the target antigen; a cleavable moiety (CM) which can be cleaved in the tumour microenvironment, cleavage of CM results in the removable of the masking moiety allowing the antibody or fragment to interact with the target antigen; an extension moiety (EX); and a linking moiety which allows covalent attachment of the shield to an antibody or fragment thereof via covalent attachment to the CH1 domain, CL domain, hinge domain and / or Fc domain of the antibody or fragment thereof. As the shield is attached via conjugation to an amino acid side chain or glycan, the present inventors have developed a platform approach to producing activatable binding molecules which can be applied to any antibody, without the need to genetically engineer the antibody.

[0009] An aspect of the invention relates to an activatable binding molecule, comprising the structure of formula (I) or (I’): MM-CM-EX-LM-AB (I)

[0010] MM-EX-CM-LM-AB (I’)

[0011] wherein AB represents an antibody or antigen binding fragment thereof capable of binding a target when the binding molecule is in an activated state;

[0012] MM represents a masking moiety capable of reducing or inhibiting interaction of AB with a target;

[0013] CM represent a cleavable moiety;

[0014] EX represents an extension moiety;

[0015] LM represents a linking moiety wherein the linking moiety is covalently attached via the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

[0016] An aspect of the invention relates to a pharmaceutical composition comprising an activatable binding molecule according to the invention and a pharmaceutical carrier.

[0017] An aspect of the invention relates to an activatable binding molecule according to the invention, or a pharmaceutical composition according to the invention for use as a medicament.

[0018] An aspect of the invention relates to an activatable binding molecule according to the invention, or a pharmaceutical composition according to the invention for use in the treatment or diagnosis of a cancer or an inflammatory disease.

[0019] An aspect of the invention relates to a method for treating a cancer comprising administering a therapeutically effective amount of an activatable binding molecule according to the invention, or a pharmaceutical composition according to the invention.

[0020] An aspect of the invention relates to a method of preparing an activatable binding molecule comprising:

[0021] conjugating a shield moiety comprising MM-CM-EX-LM- to an antibody or fragment thereof (AB), to form an activatable binding molecule,

[0022] wherein the shield moiety is conjugated to AB via the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

[0023] An aspect of the invention relates to a method of screening masking moieties (MM) comprising:

[0024] preparing one or more variant MM;

[0025] preparing a library of one or more shield moieties to be screened, wherein a shield moiety comprises MM-CM-CS1;

[0026] preparing an antibody or fragment comprising AB-LM-CS2; conjugating AB-LM-CS2 to one or more of MM-CM-CS1; and

[0027] determining the reduction or inhibition on AB binding elicited by said shield moiety; wherein MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;

[0028] CM represents a cleavable moiety;

[0029] EX represents an extension moiety;

[0030] LM represents a linking moiety;

[0031] CS1 represents one part of a conjugation system;

[0032] CS2 represents the second complimentary part of said conjugation system; and AB represents an antibody or antigen binding fragment thereof.

[0033] Figures

[0034] Figure 1. Selective target of LGR5+ cells a-LHR5-ADC. A. a-LGR5-ADC has higher activity towards pre-B-ALL cells expressing high levels of LGR5 (NALM6) versus the lower LGR5 expressing REH cells. “NC” refers to the non-cleavable form of a-LGR5-ADC that displays no killing activity. B. Organoid lines expressing higher levels of LGR5 protein are more sensitive to killing with a-LGR5-ADC treatment. C. a-LGR5-ADC displays in vivo efficacy in targeting tumours in a murine xenograft model of human pre-B-ALL.

[0035] Figure 2. The Shield Technology. A. Format of the a-LGR5-ADC-Shield. The composition of the Shield arm is shown and consists of the Spycatcher protein. MMP1 / 2 / 9 cleavage sites and a TEV cleavage site (for proof-in-concept studies) that are linked to the a-LGR5-blocking epitope peptide. B. Shielded a-LGR5 uptake by LGR5+ cells is attenuated until unmasking is triggered by Shield arm proteolysis by TEV protease.

[0036] Figure 3. SDS-PAGE gel of the collection of shield arms tested for production and cleavability. Lanes contain the various shield arms in the absence or presence of TEV protease, noted underneath the gel, except for lane 13 which contains TEV protease alone. Mass of molecule weight markers are shown on the right side of the gel. In all cases, the intact shields, denoted by * on the right side of the gel is cleaved to the lower molecular mass bands, marked as **. *** indicates the bands corresponding to the TEV protease. The shielding peptide used for anti-LGR5 was its own epitope on the LGR5 protein; for Trastuzumab, the shielding peptide was the H98 mimotope which mimics the 3-dimensional epitope for Trastuzumab. In total we generated 3 shield arms of different compositions for each of anti-LGR5 and Tras labelled LGR5_1, LGR5_2 and LGR5_3, and H98_1, H98_2 and H98_3.

[0037] Figure 4. Example of protease-mediated de-shielding. Lane 1 - Unshielded Trastuzumab (Tras). Lane 3 - shielded Tras. Lane 4 - shielded Tras cleaved with the TEV protease - * TEV protease, **cleaved small product containing shield arm C-terminus, and *** antibody moiety containing residual N terminus of the shield arm, note the shift downward that we use as our marker for TEV-mediated de-shielding of Tras. Figure 5. The shield platform can be applied to different therapeutic antibodies. Gel showing generation of shielded anti-LGR5 and shielded Tras and cleavage with TEV protease. All shielded antibodies in lanes 2-7 and 9-14 were purified by specific absorption of the 6xHis tag attached to the shield arms to Ni / NTA agarose and specific elution from the resin. Lanes 1 and 8 - anti-LGR5 and Tras with no shield. Lanes 3, 5, and 7 - shield arms have been cleaved with TEV protease. The characteristic indicator of shield cleavage for Tras is shown by *. For cleavage of shielded LGR5, we were unable to distinguish a characteristic indicator and require mass spectrometry to quantify level of shield arm cleavage. In total we generated 3 shield arms of different compositions for each of anti-LGR5 and Tras labelled LGR5_1, LGR5_2 and LGR5_3, and H98_1, H98_2 and H98_3.

[0038] Detailed Description of the Invention

[0039] 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.

[0040] 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, Kontermann and Dubel, eds., Springer-Verlag, Heidelberg (2010).

[0041] 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. Activatable Binding Molecule

[0042] An aspect of the invention relates to an activatable binding molecule comprising the structure of formula (I) or (I’):

[0043] MM-CM-EX-LM-AB (I)

[0044] MM-EX-CM-LM-AB (I’)

[0045] wherein AB represents an antibody or fragment thereof capable of binding a target when the binding molecule is in an activated state;

[0046] MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;

[0047] CM represent a cleavable moiety;

[0048] EX represents an extension moiety;

[0049] LM represents a linking moiety wherein the linking moiety is covalently attached via in the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

[0050] As shown in formula I and I’ the positioning of the CM and EX may be interchanged. In formula I and I’ the CM and EX may have any of the characteristics herein described.

[0051] Masking moiety

[0052] The term “masking moiety” or “MM” as used herein refers to a molecule that when positioned in proximity to the AB, in particular the variable region or CDRs of the AB, reduces or inhibits binding of the AB to the target antigen.

[0053] The MM may be selected for use with a specific antibody or fragment thereof. The MM may be a peptide and / or glycan sequence which is capable of reducing or inhibiting binding of the AB to the target antigen. In an embodiment the MM comprises an amino acid sequence that binds to AB and inhibits the interaction of AB with the target. In an embodiment the MM comprises an amino acid sequence that binds to the antigen binding region of AB and inhibits the interaction of AB with the target.

[0054] In certain embodiments, the MM comprises a natural binding partner of AB, for example MM may comprise an epitope of the AB. For use as a MM the structure of the epitope may be altered to modulate the binding of the AB to the MM. In an embodiment the MM comprises an epitope with one or more amino acid substitutions compared to the wild-type sequence of said epitope. In some embodiments the MM comprises high homology or similarity to a natural binding partner of the AB. In some embodiments MM may comprise more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or similarity to any natural binding partner of the AB. In some embodiments MM may comprise more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any natural binding partner of the AB.

[0055] It may be advantageous to modulate / tune the interaction of the MM to the AB, to ensure that the MM will no longer interfere with the binding of AB to the target antigen once cleavage has occurred.

[0056] In some embodiments, the MM has been selected to be substantially different to a natural binding partner of the AB. In an embodiment MM contains no or substantially no homology to any natural binding partner of the AB. In some embodiments the MM comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% homology or similarity to any natural binding partner of the AB. In some embodiments, the MM comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identity to any natural binding partner of the AB.

[0057] The MM may comprise an amino acid sequence. The amino acid sequence may have a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a length in the range of 30-40 amino acids, a length in the range of 35-40 amino acids, a length in the range of 10-30 amino acids, a length in the range of 15-30 amino acids, a length in the range of 20-30 amino acids, a length in the range of 25-30 amino acids, a length in the range of 10-20 amino acids, a length in the range of 10-15 amino acids, or a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0058] The masking moiety may reduce or inhibit binding of the AB to the target antigen via any reasonable mechanism. In some embodiments, as described above the MM directly binds to the antigen binding region of AB. In other embodiments MM sterically hinders interaction of the AB with the target antigen.

[0059] Cleavable moiety

[0060] The cleavable moiety (CM) provides a mechanism of specific activation of the AB in the tumour microenvironment. The CM comprises a moiety that it cleavable under one or more condition present in the tumour microenvironment which is distinct from healthy tissue. The condition in the tumour microenvironment may be naturally occurring or may be externally induced. In an embodiment the CL may be enzymatically cleavable, acid labile, photolabile.

[0061] The CM may be protease cleavable for example the CM may comprise an amino acid sequence that acts as a substrate for a protease. In an embodiment the CM may comprise an amino acid sequence that acts as a substrate for a protease that is upregulated in the tumour microenvironment. In an embodiment the protease is selected from a metalloprotease, a threonine protease, a serine protease, an aspartic acid protease, or a cysteine protease.

[0062] Metalloproteases are a family of Zn2+ binding protease homologues with numerous cancer-related functions and represent the largest protease group in humans. Metalloproteases exhibit diverse roles, functions, and patterns of localization, and are instrumental for pericellular proteolysis with direct effects on ECM structure, function, and signalling. Various metalloproteases have been identified that are highly active in the tumor microenvironment including matrix metalloproteases (MMPs), disintegrin and metalloproteases (ADAMs), and disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs). In an embodiment CM may comprise an amino acid sequence that acts as a substrate for an MMP, an ADAM or an ADAMTS. MMPs are categorised into 5 groups. The first category of MMP is non-furin regulated MMPs, this includes MMP-1, -3, -7, -8, -10, -12, -13, -20, and -27. The second category of MMP is MMPs consisting of three fibronectin-like inserts in the catalytic domain, this includes MMP-2 and -9. The third category of MMP is MMPs anchored to the cellular membrane by a C-terminal glycophosphatidylinositol, this includes MMP-11, -17, and -25. The fourth category of MMP is MMPs with a transmembrane domain, this includes MMP -14, -15, -16, -24. The fifth category of MMP includes all non-classified MMPs, this includes MMP -19, -21, -23, -26, and -28. In an embodiment the CM may comprise an amino acid sequence that acts as a substrate for any aforementioned MMP. In an embodiment the CM may comprise an amino acid sequence that acts as a substrate for MMP1, MMP2, and / or MMP9.

[0063] In an embodiment the CM may comprise an amino acid sequence that acts as a substrate for a cathepsin. The cathepsin may be selected from cathepsin B, cathepsin L, cathepsin S, cathepsin D. In an embodiment the CM may comprise an amino acid sequence that acts as a substrate for a kallikrein. In an embodiment the CM may comprise an amino acid sequence that acts as a substrate for Granzyme B.

[0064] Extension moiety

[0065] The present shield technology works by attaching a shield arm to an antibody. The shield arm is covalently attached to the antibody or fragment thereof (AB) via the CH1 domain, CL domain, hinge domain and / or Fc domain of AB. As such, the shield attachment site is positioned away from the N-terminal of the variable domain and the antigen binding domain. Without wishing to be bound by theory it is hypothesised that positioning the attachment site away from the antigen binding domain means that once the MM has been cleaved off any remaining portion of the shield arm will not interfere with binding.

[0066] In order to have the attachment site of the shield arm positioned away from the antigen binding site, an extension moiety (EX) is required between the LM and CM. The EX provides the necessary flexibility and length to position the MM to interact with the antigen binding site of AB.

[0067] In an embodiment the EX comprises any suitable linker to position the MM to interact with the antigen binding site of AB. For example, the EX may comprise a PEG linker, polysarcosine linker and / or a Gly / Ser linker. The skilled person will be able to select and optimise a suitable linker length for the EX. In an embodiment the EX is non-antigenic.

[0068] In certain embodiments the EX may comprise functionality which allows screening of masking moieties e.g. for use in screening methods to optimise MM characteristics. In certain embodiments the EX may comprise a conjugation system. The conjugation system may be designed to allow conjugation of MM-CM to LM-AB and form MM-CM-EX-LM-AB. In embodiments wherein a conjugation system is present one part of the conjugation system may be linked to CM and one part of the conjugation system may be linked to LM, e.g. MM-CM-CS1 and CS2-LM-AB, wherein CS1 is one part of the conjugation system and CS2 is the second part of the conjugation system. Upon conjugation of CS1 to CS2 the EX is formed. By including a conjugation system in the extension moiety this provides a modular system to screen various masking moieties for optimal characteristics. In this embodiment it is possible to develop multiple MM-CM-CS1 molecules comprising variant MM moieties, which can then be attached to the CS2-LM-AB via the conjugation system, which can then be screened for optimal masking characteristics i.e. the ability to interfere with binding of AB to the target antigen. In this way multiple variant masking moieties can be quickly and easily screened for optimal characteristics.

[0069] In an embodiment the conjugation system comprises a catcher-tag system. The catcher-tag system may be selected from SpyCatcher-SpyTag system, SnoopTag-SnoopCatcher system, Spy-Stapler-SpyTag system, Spyligase-SpyTag system, SdyCatcher-SpyTag system, SilkCatcher-SilkTag, N1 / 2-C1 / 2 system, N4 / 5-C4 / 5 system, N5 / 6-C5 / 6 system, N6 / 7b-C6 / 7b system, CnaB2Ctacher-CnaB2Tag system, PilinC-lsopeptagC system, PilinN-lsopeptagN system, DogCatcher-DogTag system, 4oq1C- 4oq1T system, 3kptCC- 3kptCT, Snoopligase-SnoopTagJr, Jo-In system.

[0070] In an embodiment MM-CM may be conjugated to LM-AB to form MM-CM-EX-LM-AB, via click chemistry. The term “click chemistry” refers to a group of reactions that are modular lining reactions, there are four key classes of click reactions including cycloadditions, nucleophilic ring openings, non-aldol carbonyl chemistry and carbon multiple bond additions. Thus, preferably, the polycyclic group is formed via a click chemistry reaction, suitably wherein a first ring is caused to fuse with a second ring via a click chemistry reaction. For example, the polycyclic group may be derived from the click chemistry reaction of a cyclic alkene or cyclic alkyne group with a tetrazine group, preferably a cyclooctene group or bicyclononyne group with a tetrazine group, most preferably trans-cyclooctene or bicyclo[6.1,0]non-4-yne with a tetrazine group. In an embodiment the click chemistry used to conjugate MM-CM to LM-AB the click chemistry is selected from an inverse electron demand Diels-Alder (iEDDA), a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and / or a strained-promoted azide-alkyne click chemistry (SPAAC) reaction.

[0071] Embodiments comprising a conjugation system in the EX have utility in linking EX to CM and / or LM and also in the development of activatable binding molecules. However, once a lead MM is identified for use in therapeutic purposes, it is advantageous to use an EX which does not comprise a conjugation system comprising a catcher tag system. In particular for activatable binding molecules for therapeutic purposes it is advantageous to use an EX which is non-antigenic. For example, where a non-antigenic EX is required a conjugation system comprising a click reaction may be preferable.

[0072] Linking moiety

[0073] The linking moiety LM refers to the moiety that covalently attaches the shield arm to the AB. The LM covalently attaches to the AB via the CH1 domain, the CL domain, the hinge domain and / or the Fc domain of AB. The LM may be attached covalently via an amino acid side chain and / or a glycan present in one or more of the CH1 domain, the CL domain, the hinge domain and / orthe Fc domain of AB. As described above the linking moiety advantageously attaches the shield arm at a site away from the from the N-terminal of the variable domain and the antigen binding domain of AB. In an embodiment the LM is covalently attached to AB via one or more amino acid side chain present in the CH1 domain, CL domain and / or Fc domain of AB. In an embodiment LM may be covalently attached to AB via one or more amino acid side chain present in the CH1 domain, CL domain, CH2 domain and / or CH3 domain of AB. In an embodiment the LM is covalently attached to AB via one or more glycan present in the CH1 domain, CL domain and / or Fc domain of AB. In an embodiment LM may be covalently attached to AB via one or more glycan present in the CH1 domain, CL domain, CH2 domain and / or CH3 domain of AB

[0074] The amino acid via which LM is attached may be selected from a naturally occurring amino acid, a genetically engineered amino acid or a genetically engineered unnatural amino acid.

[0075] In an embodiment the LM, is attached to AB via one or more thiol groups of cysteine residues. In an embodiment the LM may comprise an interchain linker. As used herein the term “interchain linker” refers to a linkerthat attaches to AB via at least two chains of AB. In a full-length antibody, there are two heavy chains and two light chains. Each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) 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) and a light chain constant region. The light chain constant region is comprised of one domain, CL. As such an interchain linker refers to a linker that is conjugated to at least two of the four chains present in an antibody or fragment thereof. For example, the interchain linker may by conjugated to one light chain and one heavy chain, or two heavy chains.

[0076] In an embodiment the LM may be attached to AB at one or more interchain disulfide position, preferably between 1 and 4 interchain disulfide positions, for example 1, 2, 3, or 4 interchain disulfide positions.

[0077] In an embodiment the LM comprises an interchain disulfide re-bridging linker. An “interchain disulfide re-bridging linker” refers to a linker which is capable of re-bridging a reduced interchain disulfide bond in the AB. Suitable interchain disulfide re-bridging linkers are described in W02023006782A1 and W02020 / 025108 (incorporated herein by reference)

[0078] In an embodiment LM comprises one or more re-bridging moieties of formula II:

[0079]

[0080] (II)

[0081] wherein:

[0082] ZAis a functional linking group;

[0083] Pep indicates the position where the re-bridging moiety is attached to the AB, such that the re-bridging moiety re-bridges a reduced interchain disulfide bond in the AB

[0084]

[0085] indicates the position where the re-bridging moiety is attached to a further moiety.

[0086] Suitably, the LM comprises between one and four re-bridging moieties of Formula (II). In an embodiment the LM comprises one re-bridging moiety of Formula (II). In an embodiment the LM comprises two re-bridging moieties of Formula (II). In an embodiment the LM comprises four re-bridging moieties of Formula (II). As will be described herein in more detail the LM has the advantage that it can be used to attach

[0087] one or more further moieties to the AB. In formula II

[0088]

[0089] * indicates the position where the rebridging moiety is attached to a further moiety. The further moiety may be a linker portion of the LM. The further moiety may be the shield arm e.g. -EX-CM-MM. The further moiety may be an active agent or payload. Suitably the one or more further moieties are selected from one or more shield arm and / or one or more active agents. In certain embodiments there is a linker present between the further moiety and the rebridging moiety.

[0090] It will be understood that the functional linking group ZAcan be any functional group that is capable of linking the antibody to the linker. The functional linking group ZAis thus a functional grouping that is capable of forming a covalent attachment to sulfur atoms present on the antibody. Suitably, the sulfur atoms are sulfur atoms from cysteine residues on the antibody, and more suitably, sulfur atoms from cysteine residues from reduced interchain disulfide bonds of the antibody.

[0091] Functional linking groups capable of linking the antibody to the linker in this way are well known in the art. Thus, the skilled person will be able to select suitable functional linking groups to use with the invention. Examples of suitable functional linking groups, ZA, include, for instance, those described in, for example, Xu 2021, Stieger 2021, Walsh 2019, Walsh 2020, Badescu 2014, Koniev 2018, Robinson 2017, Behrens 2015 and WO2019 / 011078.

[0092] In certain embodiments, ZAis selected from one of the functional linking groups shown below, and thus the conjugate comprises between one and four (preferably one, two or four) re-bridging moieties independently selected from one of the following groups:

[0093]

[0094] (Formula Ila) 5 (Formula lie)

[0095]

[0096] Formula (lid) (Formula He)

[0097] o o

[0098] Pep Pep

[0099] (Formula Ilf)

[0100]

[0101] (Formula llg)

[0102]

[0103] (Formula llh)

[0104] wherein:

[0105] one or two of A1, A2and A3are N and the other one or two of A1, A2and A3are CH, or all three of A1, A2and A3are N or C; a and b are integers selected from 0 or 1;

[0106] X is selected from N, NRN, O and S, wherein RNis H or C1-2 alkyl;

[0107] R1and R2are independently selected from C1-C6 alkylene and C1-C6 alkylene containing O in the backbone;

[0108] R3is selected from hydrogen or a C1-C4 alkyl;

[0109] Y1and Y2are independently absent or selected from O, NR4, C(=O), C(=O)NR4or NR5C(=O);

[0110] p and q are independently integers selected from 0 or 1;

[0111] Q is CR6, N or aryl;

[0112] R4, R5and R6are independently selected from hydrogen and C1-C4alkyl;

[0113] Pep indicates the position where the moiety is linked to the AB.

[0114] In certain embodiments, two of A1, A2and A3are N (e.g. A1and A2are N) and the other of A1, A2and A3is CH (e.g. A3is CH). Suitably, in this embodiment, integers a and b are 1.

[0115] In other embodiments, all three of A1, A2and A3are N. Suitably, in this embodiment, integers a and b are 1.

[0116] In some embodiments, one of A1, A2and A3is N (e.g. A3is N) and the other two of A1, A2and A3are CH (e.g. A1and A2are CH). Suitably, in this embodiment, integers a and b are 0.

[0117] Suitably, X is NH or O. Most suitably, X is NH.

[0118] In certain embodiments, R1and R2are independently selected from C1-C6 alkylene. Suitably, R1and R2are independently selected from C1-C4 alkylene.

[0119] In some embodiments, R3is a C1-C4 alkyl (e.g. a methyl group).

[0120] In some embodiments, Y1and Y2are independently absent or selected from O, NR4and C(=O). Suitably, Y1and Y2are independently absent.

[0121] Suitably, Q is CR6or N. In some embodiments, Q is CR6. In other embodiments, Q is N.

[0122] In some embodiments, R4, R5and R6are independently selected from hydrogen and methyl. Suitably, R4, R5and R6are each hydrogen.

[0123] In other embodiments, the conjugate comprises between one and four (preferably between two and four, more preferably between three and four, and most preferably four) re-bridging moieties of general formula (Illa), shown below:

[0124]

[0125] (Formula Illa)

[0126] wherein each of A1, A2, A3, X, Pep and

[0127]

[0128] are as defined herein.

[0129] In some embodiments, A1and A2are N.

[0130] In other embodiments, A1and A3are N.

[0131] In some embodiments, X is selected from NRN, O and S, where RNis H or C1-2 alkyl. In these embodiments, only a single further moiety (e.g. the shield arm -EX-CM-MM) can be attached is to X via a linker.

[0132] In some of these embodiments, X is NRN. X may be NH, NCH3 or NCH2CH3

[0133] In others of these embodiments, X is O.

[0134] In others of these embodiments, X is S.

[0135] In an embodiment LM comprises the moiety of formula (lllb):

[0136]

[0137] (Formula lllb)

[0138] wherein Q1is:

[0139]

[0140] where a1 = 0 to 5, b1 = 0 to 16, d = 0 to 5, d1 is 0 to 16, and b1 + d1 = 0 to 16; and YLis a functional linking moiety.

[0141] YLmay be selected from the group consisting of:

[0142] (

[0143]

[0144] b) '■C(SSO)HK'-,

[0145] In an embodiment the LM comprises the moiety of formula (lllc):

[0146] (Formula lllc)

[0147] where Q2 is:

[0148]

[0149] where a2 = 0 to 5, b2 = 0 to 16, c2 = 0 to 5, d2 is 0 to 16, and b2 + d2 = 0 to 16.

[0150] In an embodiment LM comprises the moiety of formula (IIId):

[0151]

[0152] (Formula Hid)

[0153] Where Q3is:

[0154]

[0155] wherein Q4is a single bond, or O, where Qxis such that Q4is an amino-acid residue, a dipeptide residue or a tripeptide residue, and L is a group for attachment to the extension moiety, EX.

[0156] In an embodiment L is selected from:

[0157] (a) a single bond;

[0158] (b) -C(=0)-;

[0159] (c) -NH-; and

[0160]

[0161] In an embodiment LM comprises the moiety of formula (Hie):

[0162]

[0163] (Formula Hie)

[0164] where D is the extension moiety EX.

[0165] In some embodiments, wherein one or more further moieties may be attached, X is N. In these embodiments, X can be attached to two further moieties via two covalent bonds, as shown below:

[0166]

[0167] wherein each of A1, A2, A3, Pep and are as defined herein. In these embodiments a linker may be present linking the further moieties to X.

[0168] In an embodiment the LM comprises the moiety of formula (lllf):

[0169]

[0170] (Formula lllf)

[0171] wherein Q1and YLare as defined in either claims 21 or 22;

[0172] Q5is:

[0173]

[0174] where a5 = 0 to 5, b5 = 0 to 16, c5 = 0 to 5, d5 is 0 to 16, and b5 + d5 = 0 to 16; and YL2is a functional linking moiety.

[0175] In an embodiment YL2is selected from the group consisting of:

[0176] (a)

[0177] (

[0178]

[0179] b) -C(=0)NH-

[0180] In an embodiment the LM comprises the moiety of formula (Illg):

[0181]

[0182] (Formula Illg)

[0183] wherein Q2is as defined herein in formula 11 Ic;

[0184] Q6is:

[0185] where a6 = 0 to 5, b6 = 0 to 16, c6 = 0 to 5, d6 is 0 to 16, and b6 + d6 = 0 to 16.

[0186] In an embodiment the LM comprises the moiety of formula (lllh):

[0187]

[0188] (Formula lllh)

[0189] wherein Q3is as defined herein in formula Hid;

[0190] Q7is:

[0191]

[0192]

[0193] wherein Q8is a single bond, or

[0194] , where Qxis such that Q4is an amino-acid residue, a dipeptide residue or a tripeptide residue, and L2is a group for attachment to an active agent or EX.

[0195] In an embodiment L2is selected from:

[0196] (a) a single bond;

[0197] (b) -C(=0)-;

[0198] (c) -NH-; and

[0199]

[0200] (d)

[0201] In an embodiment the LM is of formula (IIIi):

[0202]

[0203] (Formula IIIi)

[0204] where D is the active agent and D2 is the extension moiety EX.

[0205] Where LM comprises one of formula llla-i, at least two distinct shield arms may be attached to AB. Each distinct shield arm may comprise -LM-EX-CM-MM, as such the activatable binding molecule may comprise MM-CM-EX-LM-AB-LM-EX-CM-MM. In this manner there is a distinct shield arm for each antigen binding region of AB.

[0206] In embodiments wherein the LM comprises two or more re-bridging moieties, the LM may comprise a linker between re-bridging moieties. The linker may be such that at least two of the re-bridging moieties (i.e. the groups of Formula (II)) are separated by between 10 and 60 atoms. Suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (II)) are separated by between 10 and 50 atoms. More suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (II)) are separated by between 10 and 40 atoms. Even more suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (II)) are separated by between 12 and 40 atoms. Still more suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (II)) are separated by between 15 and 40 atoms. Most suitably, the linker is such that at least two of the re-bridging moieties (i.e. the groups of Formula (II)) are separated by between 16 and 38 atoms.

[0207] In some embodiments, the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue, an arylene-containing moiety, a heteroarylene-containing moiety, a heterocyclyl-containing moiety, a cycloalkyl-containing moiety and combinations thereof. Suitably, the linker comprises between 1 and 20 of the above-mentioned groups. More suitably, the linker comprises between 1 and 15 of the above-mentioned groups. Even more suitably, the linker comprises between 1 and 10 of the above-mentioned groups.

[0208] Suitably, the linker comprises one or more groups selected from an alkylenediamine moiety, a polyethylene glycol moiety, an amino acid residue and combinations thereof.

[0209] The alkylenediamine moiety will be understood to be a functional grouping that contains or is derived from an alkylenediamine moiety, such as, an ethylenediamine.

[0210] The polyethylene glycol moiety will be understood to be a functional grouping that comprises one or more alkylene glycol moieties, such as, one or more ethylene glycol moieties. In certain embodiments, the linker comprises between 1 and 10 alkylene glycol moieties (e.g. ethylene glycol moieties).

[0211] The amino acid residues present in the linker, will be understood to constitute both natural and unnatural amino acid residues. Suitably, the amino acid residue will be a natural amino acid residue. The amino acid residue may also comprise two or more amino acids, such as, for example, dipeptide and tripeptide moieties. Suitable amino acid residues include Phe, Lys, Vai, Ala, Cit, Leu, He, Arg, Gly, Ser and Trp residues. The arylene-containing moiety will be understood to be a functional grouping that comprises one or more arylene groups. Suitably, the arylene will be a 6-membered arylene such as phenylene. In certain embodiments, the arylene-containing moiety is a functional grouping that includes a 1,3,5-benzyl group.

[0212] The heteroarylene-containing moiety will be understood to be a functional grouping that comprises one or more heteroarylene groups. Suitably, the heteroarylene will be a functional grouping that includes a 6-membered heteroaryl such as triazine. In certain embodiments, the arylene-containing moiety is a functional grouping that includes a 1,3,5 triazine group.

[0213] The heterocyclyl-containing moiety will be understood to be a functional grouping that comprises one or more heterocycle groups. Suitably, the heterocycle is a 6-membered heterocycle such as triazinane. In certain embodiments, the heterocyclyl-containing moiety is a functional grouping that includes a 1,3,5 triazinane group.

[0214] The cycloalkyl-containing moiety will be understood to be a functional grouping that comprises one or more cycloalkyl groups. Suitably, the cycloalkyl is a 6-membered cycloalkyl such as cyclohexane. In certain embodiments, the cycloalkyl-containing moiety is a functional grouping that includes a 1,3,5 cyclohexane.

[0215] In embodiments where LM comprises two or more re-bridging moieties, the LM can provide the attachment point for one or more shield arm. In a particular embodiment the activatable binding molecule may comprise:

[0216] EX-CM-MM

[0217] I AB-LM

[0218] \

[0219] EX-CM-MM

[0220] In embodiments where LM comprises two or more re-bridging moieties, the LM can provide the attachment point for one or more shield arm and one or more further moieties e.g. an active agent.

[0221] In certain embodiments, the LM comprises a compound of Formula (IVA), shown below: Z1

[0222] Q1

[0223] Z1- Q1- Q2- Q3- (W1)r- (W2), - Q3- Q2- Q1- Z1

[0224] Q1

[0225]

[0226] (Formula IVA)

[0227] wherein:

[0228] each Z1is a re-bridging moiety of Formula (II) as defined hereinabove that is attached to an antibody at the positions shown in Formula (II);

[0229] each Q1is independently a bond or a group of Formula Va:

[0230]

[0231] Formula Va

[0232] wherein:

[0233] Q1Ais absent or selected from a C(=O), OC(=O), NR7C(=O), C(=O)NR7, C(=NR8) and C(=S);

[0234] Q1Bis selected from O, N, S and CR9CR10;

[0235] R7, R8, R9and R10are independently selected from hydrogen and C1-C4alkyl;

[0236] R20and R21are independently selected from a bond, C1-C10 alkylene and C1-C10 alkylene containing O in the backbone; and v is an integer selected from 0, 1 or 2;

[0237] each Q2is independently selected from N and CR11, wherein R11is selected from hydrogen and C1-C4alkyl;

[0238] each Q3is independently absent or a group of Formula Vb:

[0239]

[0240] Formula Vb

[0241] wherein: Q3Ais selected from NR12, O and S;

[0242] Q3Band Q3Care independently selected from a bond, O and NR13; R12and R13are independently selected from hydrogen and C1-C4alkyl; R22and R23are independently selected from a bond, C1-C10alkylene and C1-C10alkylene containing O in the backbone;

[0243] Q3Dis absent or selected from C(=O), OC(=O), NR12C(=O) and C(=O)NR12;

[0244]

[0245] Formula Vc Formula Ve wherein:

[0246] W1Ais selected from N and CR14, wherein R14is a selected from hydrogen and C1-C4alkyl;

[0247] Lwis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing amido, O or N in the backbone;

[0248] Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl or a 6-membered cycloalkyl, wherein each Xi is independently selected from CH or N;

[0249] W1Bis a group selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - OC(=O)O-, -NR15C(=O)-, -C(=O)NR15-, -NR15C(=O)NR16-, -C(=NR15)- and -C(=S)-;

[0250] R15and R16are independently selected from hydrogen and C1-C4alkyl; R23and R24are independently selected from a bond, Ci-Cwalkylene and Ci-Cwalkylene containing O in the backbone;

[0251] m is an integer selected from 1 or 2;

[0252] W1Cis selected from Formula W01or Formula W02: D

[0253] W02

[0254] L1

[0255] FG'

[0256]

[0257] Formula W01Formula W02

[0258] wherein:

[0259] W01is a group of Formula W03:

[0260]

[0261] Formula W03

[0262] wherein:

[0263] QW1is absent or selected from a C1-C12alkylene and a C1-C12alkylene containing O or N in the backbone; and

[0264] QW2is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - NHC(=O)-, and -C(=O)NH-;

[0265] y is an integer selected from 0 or 1;

[0266] XW1is selected from O or NH;

[0267] XW2is selected from hydrogen, C1-C4alkyl, ORx1and NRx1Rx2, wherein Rx1and Rx2are independently selected from hydrogen and C1-C4alkyl;

[0268] WQ2

[0269]

[0270] indicates the position where XW1is attached to a group of formula W02; R24

[0271]

[0272] indicates the position where W01is attached to a group of formula R24

[0273] W02is a group of Formula WC4:

[0274] QW1A

[0275]

[0276] Formula WC4

[0277] wherein:

[0278] QW1Ais absent or selected from a C1-C12alkylene and a C1-C12alkylene containing O or N in the backbone; and

[0279] QW2Ais selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - NHC(=O)-, and -C(=O)NH-; LQ1is a linker comprising one or more groups selected from a C1-C20alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereof;

[0280] XW3is selected from O or NH;

[0281] W

[0282]

[0283] indicates the position where XW4is attached to another group of formula W02via the QW2Asubstituent group, or V\A

[0284]

[0285] indicates the position where XW4is attached to one of the following groups: a hydrogen or - C(=O)RX3, wherein Rx3is selected from hydrogen and C1-C4alkyl;

[0286] W31

[0287]

[0288] indicates the position where QW2Ais attached to the group of formula W01via the XW1group;

[0289] XW4is selected from O or NH;

[0290] LQ1is a linker comprising one or more groups selected from a C1-C20alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereof;

[0291] t is an integer selected from 1 to 8;

[0292] FG’ is a functional linking moiety;

[0293] L1is a linker; and

[0294] D is a further moiety selected from EX and / or an active agent;

[0295] W2is a group of Formula IVd:

[0296]

[0297] Formula IVd

[0298] wherein:

[0299] W2Ais selected from N and CR17;

[0300] W2Bis a group selected from N, CR18, -C(=O)N- and -C(=O)CR18-; W2Cis a group selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - OC(=O)O-, -NR17C(=O)-, -C(=O)NR17-, -NR17C(=O)NR18-, -C(=NR17)- and -C(=S)-;

[0301] L2Wis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing amido, O or N in the backbone;

[0302] R17and R18are independently selected from hydrogen and C1-C4alkyl; R25, R26and R27are independently selected from a bond, Ci- Ci2alkylene and Ci-Ci2alkylene containing O in the backbone; and FG’, L1and D are as defined above;

[0303] r is an integer selected from 0 to 12;

[0304] s is an integer selected from 0 to 6; and

[0305] wherein r and s are selected such that the total number of further moieties per LM is between 1 and 12.

[0306]

[0307] It will be understood that when integer v is 2 the twovgroups may be the same or different.

[0308] It will also be understood that when integer m is 2 the two WB1and two R24groups may be the same or different. Suitably, the two WB1and two R24groups are the same.

[0309] Suitably, m is 1.

[0310] Suitably, Ring A is selected from a phenyl, 1,3,5- triazine or 1,3,5 triazinane.

[0311] Suitably, W1is a group of Formula Vc.

[0312] In certain embodiments, W1is a group of Formula Vc and W1Cis a group of Formula W01.

[0313] In other embodiments, W1is a group of Formula Vc and W1Cis a group of Formula W02.

[0314] In certain embodiments, Lwis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing one or more of an O or an N in the backbone.

[0315] When W1is a group of Formula Vc and W1Cis a group of Formula W02, suitably t is an integer selected from 1 to 6, most suitably t is an integer selected from 1 to 4.

[0316] Thus, it will be understood that the linker defined hereinabove in connection with the first aspect of the invention may take the form of Formula LX1or Formula LX2shown below: Z1

[0317]

[0318] Formula LX1 FG'

[0319] R24W10

[0320] Qi - Q2- Q3_ L™ - Q3.

[0321] R25W2CR27

[0322] Formula LX2wherein each of Z1, Q1, Q2, Q3, W1A, W1B, W1C, W2A, W2B, W2C, WQ1, WQ2, R23, R24, R25, R26, R27,

[0323]

[0324] FG’, L1, Lw, L2W, LQ1, m, t, r and s are as defined herein; and indicates the position where the linker is attached to the further moieties. In certain embodiments, FG’ may be a functional group formed via click reaction e.g. a cycloaddition, nucleophilic ring opening, non-aldol carbonyl chemistry and carbon multiple bond additions. In an embodiment the click reaction is selected from an inverse electron demand Diels-Alder (iEDDA), a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and / or a strained-promoted azide-alkyne click chemistry (SPAAC) reaction. In embodiments FG’ is a bond or a functional group formed from the reaction between: i) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; ii) an azide and an alkyne; iii) a amine and an acyl halide or carboxylic acid; iv) electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine); and iii) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine. FG’ may be formed from attachment of -EX-CM-MM. FG’ may be formed from attachment of a further moiety e.g. an active agent. Suitably, FG’ is a bond or a functional group formed from the reaction between an azide and an alkyne or an amine and an acyl halide or carboxylic acid. Most suitably, FG’ is a functional group formed from the reaction between an azide and an alkyne.

[0325] In embodiment the one or more further moiety may be conjugated to the LM via a reaction between: i) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; ii) an azide and an alkyne; iii) a amine and an acyl halide or carboxylic acid; iv) electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine); and iii) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine. In embodiments where both a shield arm and an active agent are attached to LM a different click reaction may be used to separately attach the shield arm and the active agent

[0326] The LM may preferably be selected from DVP (divinylpyrimidine), bis-DVP or tetra-DVP, or a derivative thereof as described in Dannheim et al Chem Sci 2022, 13, 8781 (incorporated herein by reference).

[0327] As described above the LM described herein can advantageously be conjugated to both the shield arm and a payload / active agent thereby forming an immunoconjugate. The term “active agent” and “payload” are used interchangeably herein. This approach as advantages for immunoconjugates as it provides a single attachment site on the antibody or fragment thereof for both the shield arm and the payload. As such, in some embodiments the LM can advantageously attach both the MM-CM-EX- and a payload to the AB.

[0328] 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. 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). The label may also be a biotin tag, derived from biotin. The label may also be a radioisotope or a radioisotope containing moiety. Suitably, the label is a positron emission tomography (PET) tracer. Suitable PET tracers include, for example, [18F] Fludeoxyglucose (18F) (FDG)-glucose analogue, [11 C] acetate, [11 C] methionine, [11 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).

[0329] In one embodiment, the payload may be a cell killing agent. In one embodiment, the payload may be a macrophage class switching agent. In one embodiment, the payload may be an immune-modulating payload. In one embodiment, the payload may be a light activatable pay load.

[0330] 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).

[0331]

[0187] 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.

[0332] Another example is a macrophage class switching agent.

[0333] 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. 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, orvii) a DNA-damaging payload.

[0334] Preferably the cytotoxin is a biologically active cytotoxic material. The cytotoxin may be selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines (in particular pyrrolobenzodiazepine dimers), camptothecin analogues, topoisomerase inhibitors and doxorubicin.

[0335] 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>I, <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 (TARCEVA(R), Genentech / OSI Pharm.), Bortezomib (VELCADE(R), Millenium Pharm.), Fulvestrant (FASLODEX(R), Astrazeneca), Sutent (SUI 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; topoisomerase inhibitor Dxd, difluoromethylornithine (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 (ELOXATIN(TM)) combined with 5-FU and leucovorin.

[0336] 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, LY117018, 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 ARIMIDEX(R) anastrozole; and anti-androgens such as flutamide, 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 aberrant 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

[0337] However, additionally or alternatively, the cytotoxin could also be selected from other known cytotoxins including ricin subunits and other peptide based cytotoxic materials.

[0338] In some embodiments, the compound 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. 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.

[0339] 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.

[0340] 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. In an embodiment the cytotoxic or cytostatic agent is an anti-neoplastic agent such as irinotecan or metabolites thereof. Suitable metabolites of irinotecan include SN-38.

[0341] 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 doublestrand DNA breaks, resulting in cell death. Calicheamicin is a naturally occurring enediyne antibiotic (A. L. Smith et al, J. Med. Chem., 1996, 39,11, 2103-2117). 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 excisiondependent 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); SG3199; SG2000; and SG2285.

[0342] 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.

[0343] 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.

[0344] In an embodiment, the cytotoxic or cytostatic agent is a Bis-(benzimidazole) carrier. Preferably, the drug is Hoechst 33258.

[0345] 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 guanine- specific 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 carmethizole analogue is selected from: Bis-(Hydroxymethyl)pyrrolizidine; and NSC 602668. In an embodiment, the ecteinascidin analogue is Ecteinascidin 743.

[0346] Adenine-specific alkylating agents are regiospecific and sequence-specific minor groove alkylators reacting at the N3 of adenines in polypyrimidines sequences.

[0347] 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.

[0348] 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.

[0349] 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.

[0350] 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.

[0351] 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; Pseudomonas exotoxin; 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). Pseudomonas exotoxin is an exotoxin produced by Pseudomonas aeruginosa which catalyzes the ADP-ribosylation and inactivation of EF2, which leads to protein synthesis inhibition and cell death. In an embodiment, the drug is amatoxin. In an embodiment, the drug is tubulysin. In an embodiment the drug is Pseudomonas exotoxin. In an embodiment, the drug is cytolysin. In an embodiment, the drug is dolabellanin. In an embodiment, the drug is epothilone.

[0352] 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-β-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.

[0353] 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.

[0354] In certain embodiments the payload may comprise a small molecule inhibitor with an anti-cancer activity. For example the small molecule inhibitor may be a Bcl-XI inhibitor, Bcl2 inhibitor, Bcl-w inhibitor, Bcr-Abl inhibitor, EGFR inhibitor VEGFR2 inhibitor, RET inhibitor, PDGFR inhibitor, FLT-3 inhibitor, KIT inhibitor, CSF-1 inhibitor, HER2 inhibitor, LCK inhibitor, B-raf inhibitor, mTOR inhibitor, c-KIT inhibitor, FGFR inhibitor, VEGFR inhibitor, HGFR inhibitor, Jak1 inhibitor, Jak2 inhibitor, VEGFR1-3 inhibitor, Src inhibitor, c. MET inhibitor, PDGFR-β inhibitor, MEK inhibitor, HSP90 inhibitor, MMP inhibitor, proteosome inhibitor, Akt inhibitor, NAMPT inhibitor. In certain embodiments the payload comprises a light-activatable payload, for example a infrared light activatable payload. Immunoconjugates comprising a near-infrared activatable payload enable binding molecule-mediated targeted delivery to achieve a high degree of tumor specificity, while using infrared light to activate the biophysical mechanism of the drug to accurately induce rapid death of cancer cells without harming the surrounding normal tissues. Suitable light-activatable payloads include IRDye700DX.

[0355] Antibody or fragment thereof

[0356] The activatable binding molecule of the invention comprises an antibody or antigen binding fragment thereof. The presently claimed shield technology inactivates the antibody or antigen binding fragment thereof by providing a masking moiety to block the interaction of the antibody or antigen binding fragment thereof with the intended target.

[0357] The term "antibody" broadly refers to any immunoglobulin (Ig) molecule, or antigen binding portion 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 mutant, variant, or derivative antibody formats are known in the art. The term “antibody” as used herein encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) and antibody fragments, provided they retain the antigen-binding ability of the antibody. The term “antibody” as used herein also encompasses, for example, a T-cell Engager for example a Bispecific T-cell Engager (BiTE). As will be appreciated by the skilled person Bispecific T-cell Engagers (BiTEs) are fusion proteins including two scFvs of different antibodies wherein one of the scFvs binds to T cells via the CD3 receptor, and the other to a tumor cell via a tumor specific molecule.

[0358] In a full-length antibody, each heavy chain is comprised of a heavy chain variable region or domain (abbreviated herein as HCVR) 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) and a light chain constant region. The light chain constant region is comprised of one domain, CL.

[0359] 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. Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG 1, IgG2, IgG 3, IgG4, IgAI and IgA2) or subclass. The term " CDR" refers to the complementarity-determining region within antibody variable sequences. 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.

[0360] The Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (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).

[0361] The system described by Kabat is used herein. The terms " Kabat numbering", " Kabat definitions" and " Kabat labelling" are used interchangeably herein. These terms, which are recognized in the art, refer to a system of numbering amino acid residues which are more variable (i.e., hypervariable) than other amino acid residues in the heavy and light chain variable regions of an antibody, or an antigen binding portion.

[0362] A chimeric antibody is a recombinant protein that contains the variable domains including the complementarity determining regions (CDRs) of an antibody derived from one species, such as a rodent antibody, while the constant domains of the antibody molecule are derived from those of a human antibody. For veterinary applications, the constant domains of the chimeric antibody may be derived from that of other species, such as a cat or dog.

[0363] A humanized 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. In certain embodiments, a limited number of framework region amino acid residues from the parent (rodent) antibody may be substituted into the human antibody framework region sequences.

[0364] The term "antigen binding site" refers to the part of the antibody or antibody fragment that comprises the area that specifically binds to an antigen. An antigen binding site may be provided by one or more antibody variable domains. Preferably, an antigen binding site is comprised within the associated VH and VL of an antibody or antibody fragment.

[0365] An antibody fragment according to the invention is a functional portion of an antibody, for example a F(ab')2, Fab, Fv, sFv and the like. The term refers thus to an antigen binding fragment and is interchangeably used with antigen binding portion of an antibody. Functional fragments of a full-length antibody retain the target specificity of a full-length antibody. Recombinant functional antibody fragments, such as Fab (Fragment, antibody), scFv (single chain variable chain fragments) and single domain antibodies (dAbs) have therefore been used to develop therapeutics as an alternative to therapeutics based on mAbs. Derivatives of antibodies are also within the scope.

[0366] scFv fragments (~25kDa) consist of the two variable domains, VHand VL. Naturally, VHand VLdomain 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).

[0367] The smallest antigen binding fragment is the single variable fragment, namely the VHor VLdomain. Binding to a light chain / heavy chain partner respectively is not required for target binding. Such fragments are used in single domain antibodies. A single domain antibody (~12 to 15 kDa) therefore consists of or comprises either the VHor VLdomain.

[0368] As used herein, the term "homology" generally refers to the percentage of amino acid residues in a sequence that are identical with the residues of the reference polypeptide with which it is compared, after aligning the sequences and in some embodiments after introducing gaps, if necessary, to achieve the maximum percent homology, and not considering any conservative substitutions as part of the sequence identity. Thus, as used herein, the percent homology between two amino acid sequences is equivalent to the percent identity between the two sequences. Neither N- or C-terminal extensions, tags or insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known. The percent identity or homology between two amino acid sequences can be determined using well known mathematical algorithms.

[0369] As used herein, when the term homology is used, this is interchangeably used with identity. Thus, when reference is made to sequence homology, these values also refer to sequence identity respectively.

[0370] Sequence identity is commonly defined with reference to the algorithm GAP (Wisconsin GCG package, Accelerys Inc, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences, maximising the number of matches and minimising the number of gaps. Generally, default parameters are used, with a gap creation penalty equalling 12 and a gap extension penalty equalling 4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Altschul et al (1997) Nucl. Acids Res. 25 3389-3402) may be used. Sequence identity may be defined using the Bioedit, ClustalW algorithm. Alignments were performed using Snapgene and based on MUSCLE (Multiple Sequence Comparison by Log-Expectation) algorithms (Edgar (2004a) Nucleic Acids Res 32: 1792-7; Edgar (2004b) BMC Bioinformatics 5: 113.).

[0371] The antibody or fragment thereof may comprise any therapeutic antibody or fragment, in particular commercially available antibodies. The antibody or fragment thereof may target a tumour associated antigen. Suitable tumour associated antigens are known in the art. In an embodiment the tumour associated antigen may be selected from HER2, CD30, NECTIN-4, TROP-2, folate receptor a, tissue factor, The present shield technology has the advantage that it can be used to shield a native antibody without requiring any genetic engineering of said antibody, i.e. the present method provides and “off-the-shelf shielding approach. As such the shield technology could be applied to any antibody, for example commercially available antibodies. In an embodiment the antibody may be selected from trastuzumab, brentuximab, mirvetuximab, tisotumab, sacituzumab, enfortumab.

[0372] The activatable binding molecule may comprise an antibody or fragment thereof that binds to an epitope or mimitope of LGR5, for example as described in WO2023 / 166318 (incorporated herein by reference) that binds to an epitope of LGR5 located within amino acids 22-37 of human LGR5 (SEQ ID NO:1).

[0373] Thus, the antibody may bind to an epitope of LGR5 which includes one or more of residues 22-37 of human LGR5 (SEQ ID NO:1). For example, the antibody may bind to an epitope of LGR5 which includes one or more, e.g. all of the following residues: G22, S23, S24, P25, R26, S27, G28, \

[0374]

[0375] j2$ |_30 |_31 p32 p35 q-36 |_|37

[0376] The antibody or fragment may bind to an epitope which comprises or consists of amino acids 22-37 of LGR5 (SEQ ID NO:1). The epitope is shown in SEQ ID NO:160.

[0377] Such an epitope is a linear epitope, as described fully below. The term “epitope” or “antigenic determinant” refers to a site on the surface of an antigen (e.g., LGR5) to which an immunoglobulin, antibody or antibody fragment 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.

[0378] 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 antibody fragment (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 antibody fragment.

[0379] The epitope was mapped by peptide mapping. In particular, the epitope was mapped by testing the reactivity of the antibody with overlapping peptide fragments and performing Western blot analysis.

[0380] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL CDR3 sequence as shown in Table 1 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one aspect, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL CDR3 sequence selected from SEQ ID NO. 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NO. 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79. In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0381] In one embodiment, the antibody has a VL CDR3 sequence comprising or consisting of an amino acid sequence selected from SEQ ID NO. 7, 13, 19, 25, 31, 37, 43, 49, 55, 61, 67, 73, 79 or a sequence having at least at least 90%, or at least 95% homology thereto.

[0382] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL CDR1 sequence as shown in Table 1 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one aspect, the invention relates to an antibody which binds LGR5, wherein the antibody comprises a VL CDR1 sequence selected from SEQ ID NO. 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NO. 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77 In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0383] In one embodiment, the antibody has a VL CDR1 sequence comprising or consisting of an amino acid sequence selected from SEQ ID NO. 5, 11, 17, 23, 29, 35, 41, 47, 53, 59, 65, 71, 77 or a sequence having at least at least 90%, or at least 95% homology thereto.

[0384] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL CDR2 sequence as shown in Table 1 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one aspect, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL CDR2 sequence selected from SEQ ID NO. 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NO. 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78. In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0385] In one embodiment, the antibody has a VL CDR2 sequence comprising or consisting of an amino acid sequence selected from SEQ ID NO. 6, 12, 18, 24, 30, 36, 42, 48, 54, 60, 66, 72, 78 or a sequence having at least at least 90%, or at least 95% homology thereto.

[0386] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH CDR3 sequence as shown in Table 1 below ora sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one aspect, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH CDR3 sequence selected from SEQ ID NO. 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NO. 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76 In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0387] In one embodiment, the antibody has a VH CDR3 sequence comprising or consisting of an amino acid sequence selected from SEQ ID NO. 4, 10, 16, 22, 28, 34, 40, 46, 52, 58, 64, 70, 76 or a sequence having at least at least 90%, or at least 95% homology thereto.

[0388] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH CDR1 sequence as shown in Table 1 below ora sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one aspect, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH CDR1 sequence selected from SEQ ID NO. 2, 8, 14, 40, 26, 32, 38, 44, 50, 56, 62, 68, 74 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NO. 2, 8, 14, 40, 26, 32, 38, 44, 50, 56, 62, 68, 74. In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0389] In one embodiment, the antibody has a VH CDR1 sequence comprising or consisting of an amino acid sequence selected from SEQ ID NO. 2, 8, 14, 40, 26, 32, 38, 44, 50, 56, 62, 68, 74 or a sequence having at least at least 90%, or at least 95% homology thereto.

[0390] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH CDR2 sequence as shown in Table 1 below ora sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH CDR2 sequence selected from SEQ ID NO. 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NO. 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75. In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0391] In one embodiment, the antibody has a VH CDR2 sequence comprising or consisting of an amino acid sequence selected from SEQ ID NO. 3, 9, 15, 21, 27, 33, 39, 45, 51, 57, 63, 69, 75 or a sequence having at least at least 90%, or at least 95% homology thereto. In one embodiment, the antibody comprises a combination of VH and VL CDR1, 2 and 3 sequences selected from the sequences in Table 1. In one embodiment, the activatable binding molecule comprises an antibody which has combinations ofVH and VL CDR1, CDR2 and CDR3 as shown for the clones in Table 1.

[0392] In one embodiment, the antibody comprises a set of VH and VL CDR1, 2 and 3 sequences selected from the sets of CDR1, 2 and 3 sequences as shown for the any of the clones in Table 1. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 2, VH CDR2 having SEQ ID No. 3, VH CDR3 having SEQ ID No. 4, VL CDR1 having SEQ ID No. 5, VL CDR2 having SEQ ID No. 6 and VL CDR3 having SEQ ID No. 7. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 8, VH CDR2 having SEQ ID No. 9, VH CDR3 having SEQ ID No.

[0393] 10, VL CDR1 having SEQ ID No. 11, VL CDR2 having SEQ ID No. 12 and VL CDR3 having SEQ ID No. 13. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 14, VH CDR2 having SEQ ID No. 15, VH CDR3 having SEQ ID No. 16, VL CDR1 having SEQ ID No.

[0394] 17, VL CDR2 having SEQ ID No. 18 and VL CDR3 having SEQ ID No. 19. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 20, VH CDR2 having SEQ ID No. 21, VH CDR3 having SEQ ID No. 22, VL CDR1 having SEQ ID No. 23, VL CDR2 having SEQ ID No. 24 and VL CDR3 having SEQ ID No. 25. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 26, VH CDR2 having SEQ ID No. 27, VH CDR3 having SEQ ID No. 28, VL CDR1 having SEQ ID No. 29, VL CDR2 having SEQ ID No. 30 and VL CDR3 having SEQ ID No.

[0395] 31. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 32, VH CDR2 having SEQ ID No. 33, VH CDR3 having SEQ ID No. 34, VL CDR1 having SEQ ID No. 35, VL CDR2 having SEQ ID No. 36 and VL CDR3 having SEQ ID No. 37. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 38, VH CDR2 having SEQ ID No. 39, VH CDR3 having SEQ ID No. 40, VL CDR1 having SEQ ID No. 41, VL CDR2 having SEQ ID No. 42 and VL CDR3 having SEQ ID No. 43. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 44, VH CDR2 having SEQ ID No. 45, VH CDR3 having SEQ ID No. 46, VL CDR1 having SEQ ID No. 47, VL CDR2 having SEQ ID No. 48 and VL CDR3 having SEQ ID No. 49. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 50, VH CDR2 having SEQ ID No. 51, VH CDR3 having SEQ ID No. 52, VL CDR1 having SEQ ID No. 53, VL CDR2 having SEQ ID No. 54 and VL CDR3 having SEQ ID No. 55. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 56, VH CDR2 having SEQ ID No. 57, VH CDR3 having SEQ ID No. 58, VL CDR1 having SEQ ID No. 59, VL CDR2 having SEQ ID No. 60 and VL CDR3 having SEQ ID No. 61. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 62, VH CDR2 having SEQ ID No. 63, VH CDR3 having SEQ ID No. 64, VL CDR1 having SEQ ID No. 65, VL CDR2 having SEQ ID No. 66 and VL CDR3 having SEQ ID No. 67. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 68, VH CDR2 having SEQ ID No. 69, VH CDR3 having SEQ ID No. 70, VL CDR1 having SEQ ID No. 71, VL CDR2 having SEQ ID No. 72 and VL CDR3 having SEQ ID No. 73. In one embodiment, the antibody comprises VH CDR1 having SEQ ID No. 74, VH CDR2 having SEQ ID No. 75, VH CDR3 having SEQ ID No.

[0396] 76, VL CDR1 having SEQ ID No. 77, VLCDR2 having SEQ ID No. 78 and VLCDR3 having SEQ ID No. 79.

[0397] In one embodiment the activatable binding molecule comprises an antibody or fragment thereof which binds to LGR5, wherein the VH of the antibody comprises the following CDR1, CDR2 and CDR3:

[0398] a) a CDR1 of SEQ ID No. 2 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 3 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 4 or a sequence with at least 90% homology thereto; or

[0399] b) a CDR1 of SEQ ID No. 8 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 9 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 10 or a sequence with at least 90% homology thereto; or

[0400] c) a CDR1 of SEQ ID No. 14 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 15 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 16 or a sequence with at least 90% homology thereto; or

[0401] d) a CDR1 of SEQ ID No. 20 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 21 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 22 or a sequence with at least 90% homology thereto; or

[0402] e) a CDR1 of SEQ ID No. 26 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 27 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 28 or a sequence with at least 90% homology thereto; or

[0403] f) a CDR1 of SEQ ID No. 32 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 33 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 34 or a sequence with at least 90% homology thereto; or

[0404] g) a CDR1 of SEQ ID No. 38 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 39 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 40 or a sequence with at least 90% homology thereto; or

[0405] h) a CDR1 of SEQ ID No. 44 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 45 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 46 or a sequence with at least 90% homology thereto; or

[0406] i) a CDR1 of SEQ ID No. 50 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 51 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 52 or a sequence with at least 90% homology thereto; or

[0407] j) a CDR1 of SEQ ID No. 56 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 57 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 58 or a sequence with at least 90% homology thereto; or k) a CDR1 of SEQ ID No. 62 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 63 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 64 or a sequence with at least 90% homology thereto; or

[0408] l) a CDR1 of SEQ ID No. 68 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 69 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 70 or a sequence with at least 90% homology thereto; or

[0409] m) a CDR1 of SEQ ID No. 74 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 75 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 76 or a sequence with at least 90% homology thereto.

[0410] In one embodiment the activatable binding molecule comprises an antibody or fragment thereof which binds to LGR5, wherein the VL of the antibody comprises the following CDR1, CDR2 and CDR3:

[0411] a) a CDR1 of SEQ ID No. 5 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 6 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 7 or a sequence with at least 90% homology thereto; or

[0412] b) a CDR1 of SEQ ID No. 11 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 12 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 13 or a sequence with at least 90% homology thereto; or

[0413] c) a CDR1 of SEQ ID No. 17 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 18 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 19 or a sequence with at least 90% homology thereto; or

[0414] d) a CDR1 of SEQ ID No. 23 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 24 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 25 or a sequence with at least 90% homology thereto; or

[0415] e) a CDR1 of SEQ ID No. 29 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 30 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 31 or a sequence with at least 90% homology thereto; or

[0416] f) a CDR1 of SEQ ID No. 35 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 36 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 37 or a sequence with at least 90% homology thereto; or

[0417] g) a CDR1 of SEQ ID No. 41 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 42 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 43 or a sequence with at least 90% homology thereto; or

[0418] h) a CDR1 of SEQ ID No. 47 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 48 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 49 or a sequence with at least 90% homology thereto; or

[0419] i) a CDR1 of SEQ ID No. 53 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 54 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 55 or a sequence with at least 90% homology thereto; or j) a CDR1 of SEQ ID No. 59 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 60 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 61 or a sequence with at least 90% homology thereto; or

[0420] k) a CDR1 of SEQ ID No. 65 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 66 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 67 or a sequence with at least 90% homology thereto; or

[0421] l) a CDR1 of SEQ ID No. 71 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 72 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 73 or a sequence with at least 90% homology thereto; or

[0422] m) a CDR1 of SEQ ID No. 77 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 78 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 79 or a sequence with at least 90% homology thereto.

[0423] Table 1 CDR sequences of antibodies of the invention

[0424] Nam VHCDR1 VHCDR2 VHCDR3 VLCDR1 VLCDR2 VLCDR3 e sequence sequence sequence sequence sequenc sequenc e e

[0425] 1 SEQ ID NO: SEQ ID NO:3 SEQ ID NO: SEQ ID SEQ ID SEQ ID 2 EIDPSDYYT 4 NO: 5 NO: 6 NO: 7 SYWMQ NYNQKFKG SLSGYVDY RASQDIRN YRSRLQ QQGNTL RLN S PPT

[0426] 2 SEQ ID NO: SEQ ID NO:9 SEQ ID NO: SEQ ID SEQ ID SEQ ID 8 EIDPSDSYT 10 NO: 11 NO: 12 NO: 13 NYWMQ NYNQKFKG SLSGYVDY RASQDISN YRSRLH QQGNSL RLN S PPT

[0427] 3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 14 15 16 NO: 17 NO: 18 NO: 19 SYWMQ EIDPSDSYS SLSGYVDY RASQDISN YRSRLQ QQGNTL NYNQKFKG RLN S PPT

[0428] 4 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 20 21 22 NO: 23 NO: 24 NO: 25 DYWMQ EIDPSDSYT SLSGSVDY RASQDISN YRSRLQ QQGNTL NYNQKFQG RLN S PPT

[0429] 12 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 26 27 28 NO: 29 NO: 30 NO: 31 DYYMN DINPNNGGT GYLFDY KSSQSLLN FASTRE QQHYST IYNQKFKG SSNQKNY S PLT LA

[0430]

[0431] 2.1 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 32 33 34 NO: 35 NO: 36 NO: 37 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRLH QQGNSL NYNQKFQG RLN S PPT

[0432] 2.2 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 38 39 40 NO: 41 NO: 42 NO: 43 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRLH QQGNSL NYNQKFQG RLN S PPT

[0433] 2.3 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 44 45 46 NO: 47 NO: 48 NO: 49 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRLH QQGNSL NYNQKFQG RLN S PPT

[0434] 2.4 SEQ ID NO: SEQ ID SEQ ID NO: SEQ ID SEQ ID SEQ ID 50 NO:51 52 NO: 53 NO: 54 NO: 55 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRRH QQGNSL NYNQKFQG RLN T PPT

[0435] 2.9 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 56 57 58 NO: 59 NO: 60 NO: 61 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRLH QQGNSL NYNQGFTG RLN S PPT

[0436] 2.11 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 62 63 64 NO: 65 NO: 66 NO: 67 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRLH QQGNSL NYNQGFTG RLN S PPT

[0437] 2.12 SEQ ID NO: SEQ ID NO: SEQ ID NO: SEQ ID SEQ ID SEQ ID 68 69 70 NO: 71 NO: 72 NO: 73 NYWMQ EIDPSDSYT SLSGYVDY RASQDISN YRSRRH QQGNSL NYNQGFTG RLN T PPT

[0438] 2.16 SEQ ID NO: SEQ ID SEQ ID NO: SEQ ID SEQ ID SEQ ID 74 NO:75 76 NO: 77 NO: 78 NO: 79 NYWMN EIDPSDSYT SLSGYVDY RASQDISN YRSRRH QQGNSL NYNQGFTG RLN T PPT

[0439]

[0440] In Table 1, the clones 1, 2, 3, 4 and 12 are mouse antibodies and clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11. 2.12 and 2.16 are humanized antibodies based on clone 2.

[0441] In embodiments in which the antibody is a humanized antibody, the antibody may comprise CDR1, CDR2 and CDR3 sequences as shown in Table 1 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 or sequences with at least 90% homology thereto. In one embodiment, the CDR1, CDR2 and CDR3 sequences as shown in Table 1 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 but include 1, 2 or 3 amino acid modifications, e.g. a substitution, deletion or insertion.

[0442] In one embodiment, the antibody provided comprise CDR1, CDR2 and CDR3 sequences as shown in Table 1 for Clone 2.4. Clone 2.4 is also referred to herein as a-LGR5v4, a-LGR5 variant 4 or human variant 4, see e.g. Table 5. In a preferred embodiment the activatable binding molecule may comprise an antibody comprising

[0443] i) a VH CDR1 of SEQ ID No. 50 or a sequence with at least 90% homology thereto, a VH CDR2 of SEQ ID No. 51 or a sequence with at least 90% homology thereto, a VH CDR3 of SEQ ID No.

[0444] 52 or a sequence with at least 90% homology thereto; and

[0445] ii) a VL CDR1 of SEQ ID No. 53 or a sequence with at least 90% homology thereto, a VL CDR2 of SEQ ID No. 54 or a sequence with at least 90% homology thereto, a VL CDR3 of SEQ ID No.

[0446] 55 or a sequence with at least 90% homology thereto.

[0447] As stated elsewhere the CDR sequences as given herein are according to Kabat. CDR sequences according to IMGT numbering for clone 2.4 are shown in SEQ ID Nos: 178-183.

[0448] In one embodiment, the activatable binding molecule comprises an antibody or fragment thereof which binds human LGR5, wherein the antibody comprises a VH sequence selected from those shown in Table 2 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VH sequence selected from SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID NOs: 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104. In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0449] In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL sequence selected from those shown in Table 2 below or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence identity thereto. In one embodiment, the activatable binding molecule comprises an antibody which binds LGR5, wherein the antibody comprises a VL sequence selected from SEQ ID Nos. 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology to one of SEQ ID Nos. 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105. In one embodiment, said sequence homology is at least 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%. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0450] The activatable binding molecule may comprise an antibody which binds to LGR5, the antibody comprising the VH and VL sequences shown in Table 2. Thus, the activatable binding molecule may comprises an antibody which bind to LGR5, and comprises:

[0451] a) a VH sequence of SEQ ID NO: 80 and a VL sequence of SEQ ID NO: 81;

[0452] b) a VH sequence of SEQ ID NO: 82 and a VL sequence of SEQ ID NO: 83;

[0453] c) a VH sequence of SEQ ID NO: 84 and a VL sequence of SEQ ID NO: 85;

[0454] d) a VH sequence of SEQ ID NO: 86 and a VL sequence of SEQ ID NO: 87;

[0455] e) a VH sequence of SEQ ID NO: 88 and a VL sequence of SEQ ID NO: 89;

[0456] f) a VH sequence of SEQ ID NO: 90 and a VL sequence of SEQ ID NO: 91;

[0457] g) a VH sequence of SEQ ID NO: 92 and a VL sequence of SEQ ID NO: 93;

[0458] h) a VH sequence of SEQ ID NO: 94 and a VL sequence of SEQ ID NO: 95;

[0459] i) a VH sequence of SEQ ID NO: 96 and a VL sequence of SEQ ID NO: 97;

[0460] j) a VH sequence of SEQ ID NO: 98 and a VL sequence of SEQ ID NO: 99;

[0461] k) a VH sequence of SEQ ID NO: 100 and a VL sequence of SEQ ID NO: 101

[0462] l) a VH sequence of SEQ ID NO: 102 and a VL sequence of SEQ ID NO: 103; or

[0463] m) a VH sequence of SEQ ID NO: 104 and a VL sequence of SEQ ID NO: 105.

[0464] In one embodiment, the antibody comprises comprise VH and VL sequences as shown in Table 2 for clone 2.4. In a preferred embodiment the activatable binding molecule may comprise an antibody comprising a VH sequence of SEQ ID NO: 96 and a VL sequence of SEQ ID NO: 97.

[0465] Table 2 VH and VL sequences of antibodies of the invention

[0466] Name VHVL

[0467] 1 SEQ ID NO: 80 SEQ ID NO: 81

[0468] QVQLQQPGAELVKPGASVKLSCKAS DIQMTQSTSSLSASLGDRVTINCRA GYTFTSYWMQWVKQRPGQGLEWIG SQDIRNRLNWYQQKPDGTVKLLISY EIDPSDYYTNYNQKFKG KATLTVDTS RSRLQSGVPSRFSGSGSGTEYSLTI SSTAYMQLSSLTSEDSAVYYCARSLS SNLEQEDIATYFCQQGNTLPPTFG GYVDYWGQGTTLTVSS GGTKLEVR

[0469] 2 SEQ ID NO: 82 SEQ ID NO: 83

[0470] QVQLQQPGAELVKPGASVKLSCKAS DIQMTQTTSSLSASLGDRVTISCRA GYTFTNYWMQWVKQRPGQGLEWIG SQDISNRLNWYQQKPDGTVKLLISY EIDPSDSYTNYNQKFKGKATLTVDTS RSRLHSGVPSRFSGSGSGTDYSLTI

[0471]

[0472] SSTAYMQLSSLTSEDSAVYYCARSLS SNLEQEDIATYFCQQGNSLPPTFG GYVDYWGQGTTLTVSS GGTKLEVR SEQ ID NO: 84 SEQ ID NO: 85 QVQLQQPGAELVKPGASVKLSCKAS DIQMTQTTSSLSASLGDRVTISCRA GYTFTSYWMQWVKQRPGQGLEWIG SQDISNRLNWYQQKPDGTVKLLISY EIDPSDSYSNYNQKFKGKATLTVDTS RSRLQSGVPSRFSGSGSGTDYSLT SSTAYMQLSSLTSEDSAVYYCARSLS ISNLEEEDIATYFCQQGNTLPPTFG GYVDYWGQGTTLTVSS GGTKLEVR SEQ ID NO: 86 SEQ ID NO: 87 QVQLQQPGAELVKPGASVKLSCKAS DIQMTQTTSSLSASLGDRVTISCRA GYTFTDYWMQWVKQRPGQGLEWIG SQDISNRLNWYQQKPDGTVKLLISY EIDPSDSYTNYNQKFQGKATLTVDTS RSRLQSGVPSRFSGSGSGTDYSLT SSTAYIQLSSLTSDDSAVYYCARSLS ITNLEQEDIATYFCQQGNTLPPTFG GSVDYWGQGTTLTVSS GGTKLEVR SEQ ID NO: 88 SEQ ID NO: 89 EVQLQQSGPELVKPGASVKISCKAS DIVMTQSPSSLAMSVGQKVTMSCK GYTFTDYYMNWVKQSHGKSLEWIG SSQSLLNSSNQKNYLAWYQQKPG DINPNNGGTIYNQKFKGKATLTVDKS QSPKLLVYFASTRESGVPDRFIGSG SSTAYMELRSLTSEDSAVYYCASGYL SGTDFTLTISSVQAEDLAAYFCQQH FDYWGPGTTLTVSS YSTPLTFGAGTKLELK SEQ ID NO: 90 SEQ ID NO: 91 QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGKAVKLLISY EIDPSDSYTNYNQKFQGRVTLTVDTS RSRLHSGVPSRFSGSGSGTDYTLTI TSTAYMELSSLRSEDTAVYYCARSLS SSLQPEDFATYFCQQGNSLPPTFG GYVDYWGQGTTVTVSS GGTKLEIK SEQ ID NO: 92 SEQ ID NO: 93 QVQLVQSGAEVKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGKAVKLLISY EIDPSDSYTNYNQKFQGRVTLTVDTS RSRLHSGVPSRFSGSGSGTDYTLTI TSTAYMELSSLRSEDTAVYYCARSLS SSLQPEDFATYYCQQGNSLPPTFG GYVDYWGQGTTVTVSS GGTKLEIK SEQ ID NO: 94 SEQ ID NO: 95 QVQLVQSGAEVKKPGASVKVSCKAS DIVMTQSPATLSLSPGERATLSCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGQAVRLLIS EIDPSDSYTNYNQKFQGRVTLTVDTS YRSRLHSGVPARFSGSGSGTDYTL TSTAYMELSSLRSEDTAVYYCARSLS TISSLEPEDFAVYFCQQGNSLPPTF GYVDYWGQGTTVTVSS GGGTKLEIK SEQ ID NO: 96 SEQ ID NO: 97

[0473]

[0474] QVQLVQSGAEVKKPGASVKVSCKAS EIVMTQSPATLSLSPGERATLSCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGQAVRLLIS EIDPSDSYTNYNQKFQGRVTLTVDTS YRSRRHTGIPARFSGSGSGTDYTL TSTAYMELSSLRSEDTAVYYCARSLS TISSLEPEDFAVYYCQQGNSLPPTF GYVDYWGQGTTVTVSS GGGTKLEIK

[0475] 2.9 SEQ ID NO: 98 SEQ ID NO: 99

[0476] QVQLVQSGSELKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGKAVKLLISY EIDPSDSYTNYNQGFTGRFVLSVDTS RSRLHSGVPSRFSGSGSGTDYTLTI VSTAYLQISSLKAEDTAVYYCARSLS SSLQPEDFATYFCQQGNSLPPTFG GYVDYWGQGTTVTVSS GGTKLEIK

[0477] 2.11 SEQ ID NO: 100 SEQ ID NO: 101

[0478] QVQLVQSGSELKKPGASVKVSCKAS DIQMTQSPSSLSASVGDRVTITCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGKAVKLLISY EIDPSDSYTNYNQGFTGRFVLSVDTS RSRLHSGVPSRFSGSGSGTDYTLTI VSTAYLQISSLKAEDTAVYYCARSLS SSLQPEDFATYYCQQGNSLPPTFG GYVDYWGQGTTVTVSS GGTKLEIK

[0479] 2.12 SEQ ID NO: 102 SEQ ID NO: 103

[0480] QVQLVQSGSELKKPGASVKVSCKAS DIVMTQSPATLSLSPGERATLSCRA GYTFTNYWMQWVRQAPGQGLEWIG SQDISNRLNWYQQKPGQAVRLLIS EIDPSDSYTNYNQGFTGRFVLSVDTS YRSRLHSGVPARFSGSGSGTDYTL VSTAYLQISSLKAEDTAVYYCARSLS TISSLEPEDFAVYFCQQGNSLPPTF GYVDYWGQGTTVTVSS GGGTKLEIK

[0481] 2.16 SEQ ID NO: 104 SEQ ID NO: 105

[0482] QVQLVQSGSELKKPGASVKVSCKAS EIVMTQSPATLSLSPGERATLSCRA GYTFTNYWMNWVRQAPGQGLEWM SQDISNRLNWYQQKPGQAVRLLIS GEIDPSDSYTNYNQGFTGRFVFSVD YRSRRHTGIPARFSGSGSGTDYTL TSVSTAYLQISSLKAEDTAVYYCARS TISSLEPEDFAVYYCQQGNSLPPTF LSGYVDYWGQGTTVTVSS GGGTKLEIK

[0483]

[0484] In Table 2, clones 1, 2, 3, 4 and 12 are murine antibodies and clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 are humanized antibodies.

[0485] In embodiments in which the antibody is a humanized antibody, the antibody may comprise VH and VL sequences as shown in Table 2 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 or sequences with at least 90% homology thereto. In one embodiment, the VH and VL sequences as shown in Table 2 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 but include 1,2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid modifications, e.g. a substitution, deletion or insertion.

[0486] The antibody may comprise a CH2 domain. The CH2 domain is preferably 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 preferably the CH2 domain of human IgG 1, lgG2, lgG3, or lgG4, more preferably the CH2 domain of human lgG1. The sequences of human IgG domains are known in the art.

[0487] The antibody 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. Preferably, the hinge region, or part thereof, is a human lgG1, lgG2, lgG3 or lgG4 hinge region, or part thereof. More preferably, the hinge region, or part thereof, is an IgG 1 hinge region, or part thereof.

[0488] The sequence of the CH3 domain is not particularly limited. Preferably, the CH3 domain is a human immunoglobulin G domain, such as a human lgG1, lgG2, lgG3, or lgG4 CH3 domain, most preferably a human lgG1 CH3 domain.

[0489] An antibody may comprise a human IgG 1, lgG2, lgG3, or lgG4 constant region. The sequences of human lgG1, lgG2, lgG3, or lgG4 CH3 domains are known in the art. The antibody may comprise a non-human IgG constant region, e.g., a rabbit lgG1 constant region.

[0490] In another embodiment, the antibody comprises or consists of a polypeptide sequence as shown for any one of the antibody clones in Table 3 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. Thus, the antibody comprises or consists of an amino acid sequence selected from the combination of VH, CH, VL and CL sequences for the clones shown in Table 3 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 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%.

[0491] Thus, the activatable binding molecule comprises may comprise an antibody which binds to LGR5, and comprises:

[0492] a) a VH sequence of SEQ ID NO: 106; a CH sequence of SEQ ID NO:107; a VL sequence of SEQ ID NO: 108; and a CL sequence of SEQ ID NO: 109;

[0493] b) a VH sequence of SEQ ID NO: 110; a CH sequence of SEQ ID NO:111; a VL sequence of SEQ ID NO: 112; and a CL sequence of SEQ ID NO: 113; c) a VH sequence of SEQ ID NO: 114; a CH sequence of SEQ ID NO:115; a VL sequence of SEQ ID NO: 116; and a CL sequence of SEQ ID NO: 117;

[0494] d) a VH sequence of SEQ ID NO: 118; a CH sequence of SEQ ID NO:119; a VL sequence of SEQ ID NO: 120; and a CL sequence of SEQ ID NO: 121;

[0495] e) a VH sequence of SEQ ID NO: 122; a CH sequence of SEQ ID NO:123; a VL sequence of SEQ ID NO: 124; and a CL sequence of SEQ ID NO: 125;

[0496] f) a VH sequence of SEQ ID NO: 126; a CH sequence of SEQ ID NO:127; a VL sequence of SEQ ID NO: 128; and a CL sequence of SEQ ID NO: 129;

[0497] g) a VH sequence of SEQ ID NO: 130; a CH sequence of SEQ ID NO:131; a VL sequence of SEQ ID NO: 132; and a CL sequence of SEQ ID NO: 133;

[0498] h) a VH sequence of SEQ ID NO: 134; a CH sequence of SEQ ID NO:135; a VL sequence of SEQ ID NO: 136; and a CL sequence of SEQ ID NO: 137;

[0499] i) a VH sequence of SEQ ID NO: 138; a CH sequence of SEQ ID NO:139; a VL sequence of SEQ ID NO: 140; and a CL sequence of SEQ ID NO: 141;

[0500] j) a VH sequence of SEQ ID NO: 142; a CH sequence of SEQ ID NO:143; a VL sequence of SEQ ID NO: 144; and a CL sequence of SEQ ID NO: 145;

[0501] k) a VH sequence of SEQ ID NO: 146; a CH sequence of SEQ ID NO:147; a VL sequence of SEQ ID NO: 148; and a CL sequence of SEQ ID NO: 149;

[0502] l) a VH sequence of SEQ ID NO: 150; a CH sequence of SEQ ID NO:151; a VL sequence of SEQ ID NO: 152; and a CL sequence of SEQ ID NO: 153; or

[0503] m) a VH sequence of SEQ ID NO: 154; a CH sequence of SEQ ID NO:155; a VL sequence of SEQ ID NO: 156; and a CL sequence of SEQ ID NO: 157.

[0504] Table 3 Full sequences of antibodies of the invention

[0505] Na Full sequence

[0506] me

[0507] VHCH VLCL

[0508] 1 SEQ ID NO: SEQ ID NO: 107 SEQ ID NO: 108 SEQ ID NO: 109 106 AKTTPPSVYPLAPGSAAQT DIQMTQSTSSL RADAAPTVSIFP QVQLQQPGA NSMVTLGCLVKGYFPEPVT SASLGDRVTIN PSSEQLTSGGA ELVKPGASVK VTWNSGSLSSGVHTFPAVL CRASQDIRNRL SVVCFLNNFYPK LSCKASGYTF QSDLYTLSSSVTVPSSTWP NWYQQKPDGT DINVKWKIDGSE TSYWMQWV SQTVTCNVAHPASSTKVDK VKLLISYRSRL RQNGVLNSWTD KQRPGQGLE KIVPRDCGCKPCICTVPEVS QSGVPSRFSG QDSKDSTYSMS WIGEIDPSDY SVFIFPPKPKDVLTITLTPKV SGSGTEYSLTI STLTLTKDEYER YTNYNQKFK TCWVDISKDDPEVQFSWF SNLEQEDIATY HNSYTCEATHKT GKATLTVDTS VDDVEVHTAQTKPREEQIN FCQQGNTLPP STSPIVKSFNRN SSTAYMQLS STFRSVSELPIMHQDWLNG TFGGGTKLEV EC SLTSEDSAVY KEFKCRVNSAAFPAPIEKTI R

[0509]

[0510] YCARSLSGY SKTKGRPKAPQVYTIPPPK

[0511] VDYWGQGTT EQMAKDKVSLTCMITNFFP LTVSS EDITVEWQWNGQPAENYK NTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLH EGLHNHHTEKSLSHSPGK SEQ ID NO: SEQ ID NO: 111 SEQ ID NO: 112 SEQ ID NO: 113 110 AKTTPPSVYPLAPGCGDTT DIQMTQTTSSL RADAAPTVSIFP QVQLQQPGA GSSVTLGCLVKGYFPESVT SASLGDRVTIS PSSEQLTSGGA ELVKPGASVK VTWNSGSLSSSVHTFPALL CRASQDISNRL SVVCFLNNFYPK LSCKASGYTF QSGLYTMSSSVTVPSSTWP NWYQQKPDGT DINVKWKIDGSE TNYWMQWV SQTVTCSVAHPASSTTVDK VKLLISYRSRL RQNGVLNSWTD KQRPGQGLE KLEPSGPISTINPCPPCKEC HSGVPSRFSG QDSKDSTYSMS WIGEIDPSDS HKCPAPNLEGGPSVFIFPP SGSGTDYSLTI STLTLTKDEYER YTNYNQKFK NIKDVLMISLTPKVTCWVD SNLEQEDIATY HNSYTCEATHKT GKATLTVDTS VSEDDPDVRISWFVNNVEV FCQQGNSLPP STSPIVKSFNRN SSTAYMQLS HTAQTQTHREDYNSTIRVV TFGGGTKLEV EC SLTSEDSAVY SALPIQHQDWMSGKEFKCK R YCARSLSGY VNNKDLPSPIERTISKIKGLV VDYWGQGTT RAPQVYILPPPAEQLSRKD LTVSS VSLTCLVVGFNPGDISVEW TSNGHTEENYKDTAPVLDS DGSYFIYSKLDIKTSKWEKT DSFSCNVRHEGLKNYYLKK TISRSPGK SEQ ID NO: SEQ ID NO: 115 SEQ ID NO: 116 SEQ ID NO: 117 114 AKTTPPSVYPLAPGSAAQT DIQMTQTTSSL RADAAPTVSIFP QVQLQQPGA NSMVTLGCLVKGYFPEPVT SASLGDRVTIS PSSEQLTSGGA ELVKPGASVK VTWNSGSLSSGVHTFPAVL CRASQDISNRL SVVCFLNNFYPK LSCKASGYTF QSDLYTLSSSVTVPSSTWP NWYQQKPDGT DINVKWKIDGSE TSYWMQWV SQTVTCNVAHPASSTKVDK VKLLISYRSRL RQNGVLNSWTD KQRPGQGLE KIVPRDCGCKPCICTVPEVS QSGVPSRFSG QDSKDSTYSMS WIGEIDPSDS SVFIFPPKPKDVLTITLTPKV SGSGTDYSLTI STLTLTKDEYER YSNYNQKFK TCWVDISKDDPEVQFSWF SNLEEEDIATY HNSYTCEATHKT GKATLTVDTS VDDVEVHTAQTKPREEQIN FCQQGNTLPP STSPIVKSFNRN SSTAYMQLS STFRSVSELPIMHQDWLNG TFGGGTKLEV EC SLTSEDSAVY KEFKCRVNSAAFPAPIEKTI R YCARSLSGY SKTKGRPKAPQVYTIPPPK EQMAKDKVSLTCMITNFFP

[0512]

[0513] VDYWGQGTT EDITVEWQWNGQPAENYK

[0514] LTVSS NTQPIMDTDGSYFVYSKLN VQKSNWEAGNTFTCSVLH EGLHNHHTEKSLSHSPGK SEQ ID NO: SEQ ID NO: 119 SEQ ID NO: 120 SEQ ID NO: 121 118 AKTTPPSVYPLAPGCGDTT DIQMTQTTSSL RADAAPTVSIFP QVQLQQPGA GSSVTLGCLVKGYFPESVT SASLGDRVTIS PSSEQLTSGGA ELVKPGASVK VTWNSGSLSSSVHTFPALL CRASQDISNRL SVVCFLNNFYPK LSCKASGYTF QSGLYTMSSSVTVPSSTWP NWYQQKPDGT DINVKWKIDGSE TDYWMQWV SQTVTCSVAHPASSTTVDK VKLLISYRSRL RQNGVLNSWTD KQRPGQGLE KLEPSGPISTINPCPPCKEC QSGVPSRFSG QDSKDSTYSMS WIGEIDPSDS HKCPAPNLEGGPSVFIFPP SGSGTDYSLTI STLTLTKDEYER YTNYNQKFQ NIKDVLMISLTPKVTCWVD TNLEQEDIATY HNSYTCEATHKT GKATLTVDTS VSEDDPDVRISWFVNNVEV FCQQGNTLPP STSPIVKSFNRN SSTAYIQLSS HTAQTQTHREDYNSTIRVV TFGGGTKLEV EC LTSDDSAVYY SALPIQHQDWMSGKEFKCK R CARSLSGSV VNNKDLPSPIERTISKIKGLV DYWGQGTTL RAPQVYILPPPAEQLSRKD TVSS VSLTCLVVGFNPGDISVEW TSNGHTEENYKDTAPVLDS DGSYFIYSKLDIKTSKWEKT DSFSCNVRHEGLKNYYLKK TISRSPGK SEQ ID NO: SEQ ID NO: 123 SEQ ID NO: 124 SEQ ID NO: 125 122 AKTTAPSVYPLAPVCGGTT DIVMTQSPSSL RADAAPTVSIFP EVQLQQSGP GSSVTLGCLVKGYFPEPVT AMSVGQKVTM PSSEQLTSGGA ELVKPGASVK LTWNSGSLSSGVHTFPALL SCKSSQSLLNS SVVCFLNNFYPK ISCKASGYTF QSGLYTLSSSVTVTSNTWP SNQKNYLAWY DINVKWKIDGSE TDYYMNWVK SQTITCNVAHPASSTKVDK QQKPGQSPKL RQNGVLNSWTD QSHGKSLEW KIEPRVPITQNPCPPLKECP LVYFASTRESG QDSKDSTYSMS IGDINPNNGG PCAAPDLLGGPSVFIFPPKI VPDRFIGSGSG STLTLTKDEYER TIYNQKFKGK KDVLMISLSPMVTCVVVDV TDFTLTISSVQ HNSYTCEATHKT ATLTVDKSSS SEDDPDVQISWFVNNVEVH AEDLAAYFCQ STSPIVKSFNRN TAYMELRSLT TAQTQTHREDYNSTLRVVS QHYSTPLTFGA EC SEDSAVYYC ALPIQHQDWMSGKEFKCKV GTKLELK ASGYLFDYW NNRALPSPIEKTISKPRGPV GPGTTLTVSS RAPQVYVLPPPAEEMTKKE FSLTCMITGFLPAEIAVDWT SNGRTEQNYKNTATVLDSD

[0515]

[0516] GSYFMYSKLRVQKSTWER

[0517] GSLFACSVVHEGLHNHLTT KTISRSLGK SEQ ID NO: SEQ ID NO: 127 SEQ ID NO: 128 SEQ ID NO: 129 126 ASTKGPSVFPLAPSSKSTS DIQMTQSPSSL RTVAAPSVFIFP QVQLVQSGA GGTAALGCLVKDYFPEPVT SASVGDRVTIT PSDEQLKSGTAS EVKKPGASV VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE KVSCKASGY QSSGLYSLSSWTVPSSSL NWYQQKPGKA AKVQWKVDNAL TFTNYWMQ GTQTYICNVNHKPSNTKVD VKLLISYRSRL QSGNSQESVTE WVRQAPGQ KKVEPKSCDKTHTCPPCPA HSGVPSRFSG QDSKDSTYSLSS GLEWIGEIDP PELLGGPSVFLFPPKPKDTL SGSGTDYTLTI TLTLSKADYEKH SDSYTNYNQ MISRTPEVTCVWDVSHED SSLQPEDFATY KVYACEVTHQG KFQGRVTLTV PEVKFNWYVDGVEVHNAK FCQQGNSLPP LSSPVTKSFNRG DTSTSTAYM TKPREEQYNSTYRWSVLT TFGGGTKLEIK EC ELSSLRSEDT VLHQDWLNGKEYKCKVSN AVYYCARSLS KALPAPIEKTISKAKGQPRE GYVDYWGQ PQVYTLPPSRDELTKNQVS GTTVTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO: SEQ ID NO: 131 SEQ ID NO: 132 SEQ ID NO: 133 130 ASTKGPSVFPLAPSSKSTS DIQMTQSPSSL RTVAAPSVFIFP QVQLVQSGA GGTAALGCLVKDYFPEPVT SASVGDRVTIT PSDEQLKSGTAS EVKKPGASV VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE KVSCKASGY QSSGLYSLSSWTVPSSSL NWYQQKPGKA AKVQWKVDNAL TFTNYWMQ GTQTYICNVNHKPSNTKVD VKLLISYRSRL QSGNSQESVTE WVRQAPGQ KKVEPKSCDKTHTCPPCPA HSGVPSRFSG QDSKDSTYSLSS GLEWIGEIDP PELLGGPSVFLFPPKPKDTL SGSGTDYTLTI TLTLSKADYEKH SDSYTNYNQ MISRTPEVTCVWDVSHED SSLQPEDFATY KVYACEVTHQG KFQGRVTLTV PEVKFNWYVDGVEVHNAK YCQQGNSLPP LSSPVTKSFNRG DTSTSTAYM TKPREEQYNSTYRWSVLT TFGGGTKLEIK EC ELSSLRSEDT VLHQDWLNGKEYKCKVSN AVYYCARSLS KALPAPIEKTISKAKGQPRE GYVDYWGQ PQVYTLPPSRDELTKNQVS GTTVTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN

[0518]

[0519] VFSCSVMHEALHNHYTQKS

[0520] LSLSPGK SEQ ID NO: SEQ ID NO: 135 SEQ ID NO: 136 SEQ ID NO: 137 134 ASTKGPSVFPLAPSSKSTS DIVMTQSPATL RTVAAPSVFIFP QVQLVQSGA GGTAALGCLVKDYFPEPVT SLSPGERATLS PSDEQLKSGTAS EVKKPGASV VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE KVSCKASGY QSSGLYSLSSWTVPSSSL NWYQQKPGQ AKVQWKVDNAL TFTNYWMQ GTQTYICNVNHKPSNTKVD AVRLLISYRSR QSGNSQESVTE WVRQAPGQ KKVEPKSCDKTHTCPPCPA LHSGVPARFS QDSKDSTYSLSS GLEWIGEIDP PELLGGPSVFLFPPKPKDTL GSGSGTDYTL TLTLSKADYEKH SDSYTNYNQ MISRTPEVTCVWDVSHED TISSLEPEDFA KVYACEVTHQG KFQGRVTLTV PEVKFNWYVDGVEVHNAK VYFCQQGNSL LSSPVTKSFNRG DTSTSTAYM TKPREEQYNSTYRWSVLT PPTFGGGTKLE EC ELSSLRSEDT VLHQDWLNGKEYKCKVSN IK AVYYCARSLS KALPAPIEKTISKAKGQPRE GYVDYWGQ PQVYTLPPSRDELTKNQVS GTTVTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO: SEQ ID NO: 139 SEQ ID NO: 140 SEQ ID NO: 141 138 ASTKGPSVFPLAPSSKSTS EIVMTQSPATL RTVAAPSVFIFP QVQLVQSGA GGTAALGCLVKDYFPEPVT SLSPGERATLS PSDEQLKSGTAS EVKKPGASV VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE KVSCKASGY QSSGLYSLSSWTVPSSSL NWYQQKPGQ AKVQWKVDNAL TFTNYWMQ GTQTYICNVNHKPSNTKVD AVRLLISYRSR QSGNSQESVTE WVRQAPGQ KKVEPKSCDKTHTCPPCPA RHTGIPARFSG QDSKDSTYSLSS GLEWIGEIDP PELLGGPSVFLFPPKPKDTL SGSGTDYTLTI TLTLSKADYEKH SDSYTNYNQ MISRTPEVTCVWDVSHED SSLEPEDFAVY KVYACEVTHQG KFQGRVTLTV PEVKFNWYVDGVEVHNAK YCQQGNSLPP LSSPVTKSFNRG DTSTSTAYM TKPREEQYNSTYRWSVLT TFGGGTKLEIK EC ELSSLRSEDT VLHQDWLNGKEYKCKVSN AVYYCARSLS KALPAPIEKTISKAKGQPRE GYVDYWGQ PQVYTLPPSRDELTKNQVS GTTVTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN

[0521]

[0522] VFSCSVMHEALHNHYTQKS

[0523] LSLSPGK SEQ ID NO: SEQ ID NO: 143 SEQ ID NO: 144 SEQ ID NO: 145 142 ASTKGPSVFPLAPSSKSTS DIQMTQSPSSL RTVAAPSVFIFP QVQLVQSGS GGTAALGCLVKDYFPEPVT SASVGDRVTIT PSDEQLKSGTAS ELKKPGASVK VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE VSCKASGYT QSSGLYSLSSWTVPSSSL NWYQQKPGKA AKVQWKVDNAL FTNYWMQW GTQTYICNVNHKPSNTKVD VKLLISYRSRL QSGNSQESVTE VRQAPGQGL KKVEPKSCDKTHTCPPCPA HSGVPSRFSG QDSKDSTYSLSS EWIGEIDPSD PELLGGPSVFLFPPKPKDTL SGSGTDYTLTI TLTLSKADYEKH SYTNYNQGF MISRTPEVTCVWDVSHED SSLQPEDFATY KVYACEVTHQG TGRFVLSVDT PEVKFNWYVDGVEVHNAK FCQQGNSLPP LSSPVTKSFNRG SVSTAYLQIS TKPREEQYNSTYRWSVLT TFGGGTKLEIK EC SLKAEDTAVY VLHQDWLNGKEYKCKVSN YCARSLSGY KALPAPIEKTISKAKGQPRE VDYWGQGTT PQVYTLPPSRDELTKNQVS VTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO: SEQ ID NO: 147 SEQ ID NO: 148 SEQ ID NO: 149 146 ASTKGPSVFPLAPSSKSTS DIVMTQSPATL RTVAAPSVFIFP QVQLVQSGS GGTAALGCLVKDYFPEPVT SLSPGERATLS PSDEQLKSGTAS ELKKPGASVK VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE VSCKASGYT QSSGLYSLSSWTVPSSSL NWYQQKPGQ AKVQWKVDNAL FTNYWMQW GTQTYICNVNHKPSNTKVD AVRLLISYRSR QSGNSQESVTE VRQAPGQGL KKVEPKSCDKTHTCPPCPA LHSGVPARFS QDSKDSTYSLSS EWIGEIDPSD PELLGGPSVFLFPPKPKDTL GSGSGTDYTL TLTLSKADYEKH SYTNYNQGF MISRTPEVTCVWDVSHED TISSLEPEDFA KVYACEVTHQG TGRFVLSVDT PEVKFNWYVDGVEVHNAK VYFCQQGNSL LSSPVTKSFNRG SVSTAYLQIS TKPREEQYNSTYRWSVLT PPTFGGGTKLE EC SLKAEDTAVY VLHQDWLNGKEYKCKVSN IK YCARSLSGY KALPAPIEKTISKAKGQPRE VDYWGQGTT PQVYTLPPSRDELTKNQVS VTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN

[0524]

[0525] VFSCSVMHEALHNHYTQKS

[0526] LSLSPGK SEQ ID NO: SEQ ID NO: 151 SEQ ID NO: 152 SEQ ID NO: 153 150 ASTKGPSVFPLAPSSKSTS EIVMTQSPATL RTVAAPSVFIFP QVQLVQSGS GGTAALGCLVKDYFPEPVT SLSPGERATLS PSDEQLKSGTAS ELKKPGASVK VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE VSCKASGYT QSSGLYSLSSWTVPSSSL NWYQQKPGQ AKVQWKVDNAL FTNYWMQW GTQTYICNVNHKPSNTKVD AVRLLISYRSR QSGNSQESVTE VRQAPGQGL KKVEPKSCDKTHTCPPCPA RHTGIPARFSG QDSKDSTYSLSS EWIGEIDPSD PELLGGPSVFLFPPKPKDTL SGSGTDYTLTI TLTLSKADYEKH SYTNYNQGF MISRTPEVTCVWDVSHED SSLEPEDFAVY KVYACEVTHQG TGRFVLSVDT PEVKFNWYVDGVEVHNAK YCQQGNSLPP LSSPVTKSFNRG SVSTAYLQIS TKPREEQYNSTYRWSVLT TFGGGTKLEIK EC SLKAEDTAVY VLHQDWLNGKEYKCKVSN YCARSLSGY KALPAPIEKTISKAKGQPRE VDYWGQGTT PQVYTLPPSRDELTKNQVS VTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN VFSCSVMHEALHNHYTQKS LSLSPGK SEQ ID NO: SEQ ID NO: 155 SEQ ID NO: 156 SEQ ID NO: 157 154 ASTKGPSVFPLAPSSKSTS EIVMTQSPATL RTVAAPSVFIFP QVQLVQSGS GGTAALGCLVKDYFPEPVT SLSPGERATLS PSDEQLKSGTAS ELKKPGASVK VSWNSGALTSGVHTFPAVL CRASQDISNRL VVCLLNNFYPRE VSCKASGYT QSSGLYSLSSWTVPSSSL NWYQQKPGQ AKVQWKVDNAL FTNYWMNW GTQTYICNVNHKPSNTKVD AVRLLISYRSR QSGNSQESVTE VRQAPGQGL KKVEPKSCDKTHTCPPCPA RHTGIPARFSG QDSKDSTYSLSS EWMGEIDPS PELLGGPSVFLFPPKPKDTL SGSGTDYTLTI TLTLSKADYEKH DSYTNYNQG MISRTPEVTCVWDVSHED SSLEPEDFAVY KVYACEVTHQG FTGRFVFSVD PEVKFNWYVDGVEVHNAK YCQQGNSLPP LSSPVTKSFNRG TSVSTAYLQI TKPREEQYNSTYRWSVLT TFGGGTKLEIK EC SSLKAEDTAV VLHQDWLNGKEYKCKVSN YYCARSLSG KALPAPIEKTISKAKGQPRE YVDYWGQGT PQVYTLPPSRDELTKNQVS TVTVSS LTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDG SFFLYSKLTVDKSRWQQGN

[0527]

[0528] VFSCSVMHEALHNHYTQKS

[0529] LSLSPGK

[0530]

[0531] In one embodiment, the antibody comprises the heavy and / or light chain as shown below or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto.

[0532] Heavy chain SEQ ID NO: 158 MPLLLLLPLLWAGALAQVQLVQSGAEVKKPGASVKVSCKASGYTFTNYWMQWVRQAPGQG LEWIGEIDPSDSYTNYNQKFQGRVTLTVDTSTSTAYMELSSLRSEDTAVYYCARSLSGYVDY WGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCP APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW QQGNVFSCSVMHEALHNHYTQKSLSLSPGK

[0533] Light chain SEQ ID NO: 159 MPLLLLLPLLWAGALAEIVMTQSPATLSLSPGERATLSCRASQDISNRLNWYQQKPGQAVRLL ISYRSRRHTGIPARFSGSGSGTDYTLTISSLEPEDFAVYYCQQGNSLPPTFGGGTKLEIKRTVA APSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

[0534] In Table 3, clones 1, 2, 3, 4 and 12 are murine antibodies and clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 are humanized antibodies.

[0535] In embodiments in which the antibody is a humanized antibody, the antibody may comprise sequences as shown in Table 3 for clones 2.1, 2.2, 2.3, 2.4, 2.9, 2.11, 2.12 and 2.16 or a sequence with at least 90% homology thereto.

[0536] In some embodiments, the activatable binding molecule comprises an antibody that is a variant of any of the above antibodies or fragments having one or more amino acid modifications, e.g. substitutions, deletions, additions, insertions or other modifications, and which retains a biological function of the antibody. Thus, variant antibodies can be sequence engineered. Modifications include at least one substitution, deletion or insertion of one or more codons encoding the antibody or polypeptide that results in a change in the amino acid sequence as compared with the native antibody or polypeptide. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and / or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence. A variant of an antibody described herein has at least 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% sequence homology to the nonvariant molecule, preferably at least 95%, 96%, 97%, 98% or 99% sequence homology.

[0537] In one embodiment, the modification is a conservative sequence modification. As used herein, the term "conservative sequence modifications" is intended to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence. Such conservative modifications include amino acid substitutions, additions and deletions. Modifications can be introduced into an antibody by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are ones in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, tryptophan), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, one or more amino acid residues within the CDR regions of an antibody of the invention can be replaced with other amino acid residues from the same side chain family and the altered antibody can be tested for retained function (i.e., the functions set forth in (c) through (I) above) using the functional assays described herein.

[0538] In some embodiments, the activatable binding molecule comprises an antibody that is a variant of an antibody selected from those shown in Table 3 that comprises one or more sequence modifications and has improvements in one or more of a property such as binding affinity, specificity, thermostability, expression level, effector function, glycosylation, reduced immunogenicity, or solubility as compared to the unmodified antibody. Sequence modifications may reside in the CDR1, CDR2 and / or CDR3 or in one or more of the framework regions.

[0539] In one embodiment, the activatable binding molecule comprises SEQ ID NO. 161, 162 or 162 or a sequence having at least 90% or 95% sequence identity thereto. These are the sequence of shield arms containing the anti-LGR5 epitope.

[0540] The activatable binding molecule may comprise an antibody or fragment thereof that binds to an epitope of HER2 protein (human epidermal growth factor receptor 2). In one embodiment, the antibody binds to an epitope of human HER2 as shown in SEQ ID NO.

[0541] 174 or a mimitope thereof.

[0542] In one embodiment, the antibody is Trastuzumab.

[0543] In one embodiment, the activatable binding molecule comprises an antibody which binds HER2, wherein the antibody comprises or consists of a VL CDR3 comprising SEQ ID NO. 173 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0544] In one embodiment, the activatable binding molecule comprises an antibody which binds HER2, wherein the antibody comprises or consists of a VL CDR1 comprising SEQ ID NO. 171 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0545] In one embodiment, the activatable binding molecule comprises an antibody which binds HER2, wherein the antibody comprises or consists of a VL CDR2 comprising SEQ ID NO. 172 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0546] In one embodiment, the activatable binding molecule comprises an antibody which binds HER2, wherein the antibody comprises or consists of a VH CDR3 comprising SEQ ID NO. 170 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0547] In one embodiment, the activatable binding molecule comprises an antibody which binds HER2, wherein the antibody comprises or consists of a VH CDR2 comprising SEQ ID NO. 169 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0548] In one embodiment, the activatable binding molecule comprises an antibody which binds HER2, wherein the antibody comprises or consists of a VH CDR1 comprising SEQ ID NO. 168 or a sequence with at least 60%, 70%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, said sequence homology is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99%.

[0549] In one embodiment, the antibody comprises a VH sequence comprising SEQ ID NO. 168 or a sequence with at least 75%, 80%, 90%, 95% or more sequence homology thereto, SEQ ID NO.

[0550] 169 or a sequence with at least 75%, 80%, 90%, 95% or more sequence homology thereto and SEQ ID NO. 170 or a sequence with at least 90% homology thereto. In one embodiment, the antibody comprises a VL sequence comprising SEQ ID NO. 171 or a sequence with at least 90% homology thereto, SEQ ID NO. 172 or a sequence with at least 75%, 80%, 90%, 95% or more sequence homology thereto and SEQ ID NO. 173 or a sequence with at least 75%, 80%, 90%, 95% or more sequence homology thereto. In one embodiment, the antibody comprises VH and VH sequences as defined in the forgoing.

[0551] In one embodiment, the antibody comprises a VH sequence comprising SEQ ID NO. 164 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. In one embodiment, the antibody comprises a VL sequence comprising SEQ ID NO. 165 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. In one embodiment, the antibody comprises VH and VH sequences as defined in the forgoing.

[0552] In one embodiment, the antibody comprises a heavy chain sequence comprising SEQ ID NO.

[0553] 166 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. In one embodiment, the antibody comprises a light chain sequence comprising SEQ ID NO. 167 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. In one embodiment, the antibody comprises heavy and light chains as defined in the forgoing.

[0554] In one embodiment, the activatable binding molecule comprises SEQ ID NO. 175, 176 or 178 or a sequence having at least 75%, 80%, 90% or 95% sequence identity thereto. These are the sequence of shield arms containing the mimitope.

[0555] An antibody as used according to the invention may comprise a human IgG Fc with effector function.

[0556] 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 FcyRI 11 are low to intermediate affinity (micromolar range Kd). In antibody dependent cellular cytotoxicity (ADCC), FcvRs on the surface of effector cells (natural killer cells, macrophages, monocytes and eosinophils) bind to the Fc region of an IgG which itself is bound to a target cell. Upon binding a signalling pathway is triggered which results in the secretion of various substances, such as lytic enzymes, perforin, granzymes and tumour necrosis factor, which mediate in the destruction of the target cell. The level of ADCC effector function various for IgG subtypes. Although this is dependent on the allotype and specific FcvR in simple terms ADCC effector function is high for human lgG1 and lgG3, and low for lgG2 and lgG4. See below for IgG subtype variation in effector functions, ranked in decreasing potency.

[0557] FcyRs bind to IgG asymmetrically across the hinge and upper CH2 region. Knowledge of the binding site has resulted in engineering efforts to modulate IgG effector functions.

[0558] The antibody may have an Fc with effector function, with enhanced effector function or with reduced effector function.

[0559] The potency of antibodies can be increased by enhancement of the ability to mediate cellular cytotoxicity functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) and antibody-dependent cell-mediated phagocytosis (ADCP). A number of mutations within the Fc domain have been identified that either directly or indirectly enhance binding of Fc receptors and significantly enhance cellular cytotoxicity: the mutations S239D / A330L / I332E (“3M”), F243L or G236A. Alternatively enhancement of effector function can be achieved by modifying the glycosylation of the Fc domain, FcyRs interact with the carbohydrates on the CH2 domain and the glycan composition has a substantial effect on effector function activity. Afucosylated (non-fucosylated) antibodies, exhibit greatly enhanced ADCC activity through increased binding to FcyRllla.

[0560] Activation of ADCC and CDC may be desirable for some therapeutic antibodies, however, in some embodiments, an antibody that does not activate effector functions is preferred. Due to their lack of effector functions, lgG4 antibodies are the preferred IgG subclass for receptor blocking without cell depletion. However, lgG4 molecules can exchange half molecules in a dynamic process termed Fab-arm exchange. This phenomenon can occur between therapeutic antibodies and endogenous lgG4. The S228P mutation has been shown to prevent this recombination process allowing the design of lgG4 antibodies with a reduced propensity for Fabarm exchange. Fc engineering approaches have been used to determine the key interaction sites for the lgG1 Fc domain with Fey receptors and C1 q and then mutate these positions to reduce or abolish binding. Through alanine scanning the binding site of C1 q to a region covering the hinge and upper CH2 of the Fc domain was identified. The CH2 domain of an antibody or fragment of the invention may comprise one or more mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors, such as FcyRI, FcyRlla, FcyRllb, FcyRIII and / or to complement. CH2 domains of human IgG domains normally bind to Fey receptors and complement, decreased binding to Fey receptors is expected to decrease antibody-dependent cell-mediated cytotoxicity (ADCC) and decreased binding to complement is expected to decrease the complement-dependent cytotoxicity (CDC) activity of the antibody molecule. Mutations to decrease or abrogate binding of the CH2 domain to one or more Fey receptors and / or complement are known in the art. An antibody may comprise an Fc with modifications K322A / L234A / L235A or L234F / L235E / P331 S (“TM”), which almost completely abolish FcyR and C1q binding. An antibody may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) (" LALA mutation"). Furthermore, complement activation and ADCC can be decreased by mutation of Pro329 (position according to EU numbering), e.g., to either P329A or P329G. The antibody may comprise a CH2 domain, wherein the CH2 domain comprises alanine residues at EU positions 234 and 235 (positions 1.3 and 1.2 by IMGT numbering) and an alanine (LALA- PA) or glycine (LALA-PG) at EU position 329 (position 114 by IMGT numbering). Additionally or alternatively an antibody may comprise an alanine, glutamine or glycine at EU position 297 (position 84.4 by IMGT numbering).

[0561] Modification of glycosylation on asparagine 297 of the Fc domain, which is known to be required for optimal FcR interaction may confer a loss of binding to FcRs; a loss of binding to FcRs has been observed in N297 point mutations. An antibody may comprise an Fc with an N297A, N297G or N297Q mutation. An antibody with an aglycosyl Fc domain may be obtained by enzymatic deglycosylation, by recombinant expression in the presence of a glycosylation inhibitor, or following the expression of Fc domains in bacteria.

[0562] IgG naturally persists for a prolonged period in the serum due to FcRn-mediated recycling, giving it a typical half-life of approximately 21 days. Half-life can be extended by engineering the pH-dependant interaction of the Fc domain with FcRn to increase affinity at pH 6.0 while retaining minimal binding at pH 7.4. The T250Q / M428L variant, conferred an approximately 2-fold increase in IgG half-life (assessed in rhesus monkeys), while the M252Y / S254T / T256E variant ('YTE”), gave an approximately 4-fold increase in IgG half-life (assessed in cynomolgus monkeys). Extending half-life may allow the possibility of decreasing administration frequency, while maintaining or improving efficacy.

[0563] The activatable binding molecule may comprise an antibody preferably having a Kd value of less than around 4 nM, less than around 3 nM, less than around 3 nM, less than around 2 nM, less than around 1 nM. The term " Kd" refers to the "equilibrium dissociation constant" and refers to the value obtained in a titration measurement at equilibrium, or by dividing the dissociation rate constant (Koff) by the association rate constant (Kon). “Ka” refers to the affinity constant. The association rate constant, the dissociation rate constant and the equilibrium dissociation constant are used to represent the binding affinity of an antibody to an antigen. Methods for determining association and dissociation rate constants are well known in the art. Using fluorescence-based techniques offers high sensitivity and the ability to examine samples in physiological buffers at equilibrium. Other experimental approaches and instruments such as a BIAcore® (biomolecular interaction analysis) assay can be used.

[0564] In embodiments, the antibody according to the invention has a Kd value in the nanomolar range.

[0565] In certain embodiments the AB is conjugated to an active agent or payload. The active agent or payload conjugated to AB may be any active agent or payload described herein. Techniques for conjugating payloads 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 AB, 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).

[0566] The payload may be conjugated to the AB 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. Alternatively or additionally, the payload may be conjugated to the AB via click chemistry as set out herein. For example, the payload may be conjugated to the first and / or second antibody or antigen-binding fragment via an inverse electron demand Diels-Alder (iEDDA) reaction.

[0567] The linkers may be attached via nucleophilic groups on the first and / or second antibody or antigen-binding fragment, 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 AB, 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.

[0568] Pharmaceutical Composition

[0569] An aspect of the present invention also relates to a pharmaceutical composition comprising the activatable binding molecule of the invention. As such, there is provided a pharmaceutical composition comprising an activatable binding molecule according to the present invention and, optionally, a pharmaceutically acceptable carrier.

[0570] An activatable binding molecule or pharmaceutical composition of the invention can be administered 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, by inhalation, or topical, particularly to the ears, nose, eyes, or skin or by inhalation.

[0571] Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intraperitoneal, intranasal, rectal, intravesical, intradermal, topical or subcutaneous administration. Preferably, the compositions are administered parenterally.

[0572] 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 an activatable binding molecule 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 coloring agents can be used. In one embodiment, when administered to an animal, the activatable binding molecule of the present invention or compositions and pharmaceutically acceptable carriers are sterile. Water is a preferred carrier when the drug antibody conjugates of the present invention are 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.

[0573] The pharmaceutical composition of the invention 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 subcutaneously.

[0574] When intended for oral administration, the composition is preferably 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.

[0575] 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.

[0576] 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.

[0577] Compositions can take the form of one or more dosage units.

[0578] In specific embodiments, it can be desirable to administer the composition locally to the area in need of treatment, or by intravenous injection or infusion. The amount of the activatable binding molecule of the present invention that is effective / active in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. 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 shall be taken into account.

[0579] Typically, the amount is at least about 0.01% of an antibody of the present invention by weight of the composition. When intended for oral administration, this amount can be varied to range from about 0.1 % to about 80% by weight of the composition. Preferred oral compositions can comprise from about 4% to about 50% of the activatable binding molecule of the present invention by weight of the composition.

[0580] Preferred compositions of the present invention are prepared so that a parenteral dosage unit contains from about 0.01 % to about 2% by weight of the activatable binding molecule of the present invention.

[0581] For administration by injection, the composition can comprise from about typically about 0.1 mg / kg to about 250 mg / kg of the animal's body weight, preferably, between about 0.1 mg / kg and about 20 mg / kg of the animal's body weight, and more preferably about 1 mg / kg to about 10 mg / kg of the animal's body weight. In one embodiment, the composition is administered at a dose of about 1 to 30 mg / kg, e.g., about 5 to 25 mg / kg, about 10 to 20 mg / kg, about 1 to 5 mg / kg, or about 3 mg / kg. The dosing schedule can vary from e.g., once a week to once every 2, 3, or 4 weeks.

[0582] Therapy

[0583] An aspect of the invention provides methods of treating disease, for example cancer, in a subject, e.g., a mammal (e.g. human patient), comprising administering an effective amount of the activatable binding molecule or pharmaceutical composition of the present invention to the subject. The method is described in detail herein.

[0584] As used herein, "treat", "treating" or "treatment" means inhibiting or relieving a disease or disorder. For example, treatment can include a postponement of development of the symptoms associated with a disease or disorder, and / or a reduction in the severity of such symptoms that will, or are expected, to develop with said disease. The terms include ameliorating existing symptoms, preventing additional symptoms, and ameliorating or preventing the underlying causes of such symptoms. Thus, the terms denote that a beneficial result is being conferred on at least some of the subjects, e.g., human patients, being treated. Many medical treatments are effective for some, but not all, patients that undergo the treatment.

[0585] The term "subject" or "patient" refers to an animal which is the object of 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.

[0586] As used herein, the term "effective amount" means an amount of an antibody, that when administered alone or in combination with an additional therapeutic agent to a cell, tissue, or subject, is effective to achieve the desired therapeutic or prophylactic effect under the conditions of administration.

[0587] The invention also relates to an activatable binding molecule or pharmaceutical composition of the invention for use in the treatment or prevention of a disease.

[0588] In another aspect, the invention relates to an activatable binding molecule or pharmaceutical composition of the invention for use in the treatment or prevention of cancer.

[0589] In another aspect, the invention relates to the use of an activatable binding molecule or pharmaceutical composition of the invention in the treatment or prevention of a disease.

[0590] In another aspect, the invention relates to the use of an activatable binding molecule or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of cancer.

[0591] The cancer may be a solid or non-solid tumor. For example, the cancer may be selected from cancer of the head or neck, uterine cancer, colorectal cancer, stomach cancer, carcinoma of the endometrium, cancer of the esophagus, leukemia, such as acute lymphoblastic leukemia (ALL), liver cancer, such as hepatocellular carcinoma or pancreatic cancer.

[0592] In one embodiment, the tumour is a solid tumour. Solid tumours which may be treated include colorectal carcinoma, for example. Some examples of such tumours include epidermoid tumours, squamous tumours, such as head and neck tumours, colorectal tumours.

[0593] In one embodiment, the tumour is a non-solid tumour. Examples of non-solid tumours include leukemia. In one embodiment, the cancer is selected from colorectal cancer (CRC), hepatocellular carcinoma (HCC), and pre-B ALL.

[0594] In one embodiment, the cancer is identified as a LGR5 positive cancer. The term “LGR5 positive cancer” as used herein, means a cancer whose cells express LGR5. The activatable binding molecule and pharmaceutical compositions of the present invention are particularly useful for the treatment of cancers that express abnormally high levels of LGR5, for example cancers which overexpress LGR5. The term “overexpress” as used herein means that the cells express more LGR5 than that observed in normal, non-cancerous cells. The cancer cells may express 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% more LGR5 than that observed in normal, non-cancerous cells. The skilled person will be well aware how to assess whether a cancer is LGR5 positive and / or overexpresses LGR5. Methods include for example, using fluorescence in-situ hybridization, immunohistochemistry approaches, flow cytometry, RT-PCR.

[0595] The present inventors have found that by utilizing the activatable binding molecule of the present invention, a toxic payload, in the form of an antibody drug conjugate (ADC) can be delivered to LGR5-expressing cancer cells to kill those cells without systemic toxicity.

[0596] In one embodiment, the cancer is locally advanced, unresectable, metastatic, or recurrent cancer.

[0597] In one embodiment, the cancer has progressed after another treatment, for example chemotherapy.

[0598] The activatable binding molecule or pharmaceutical composition of the invention may be administered as the sole active ingredient or in combination with one or more other therapeutic agents. A therapeutic agent is a compound or molecule which is useful in the treatment of a disease. Examples of therapeutic agents include antibodies, antibody fragments, drugs, toxins, nucleases, hormones, immunomodulators, pro-apoptotic agents, anti-angiogenic agents, boron compounds, photoactive agents or dyes and radioisotopes. An antibody molecule includes a full antibody or fragment thereof (e.g., a Fab, F(ab')2, Fv, a single chain Fv fragment (scFv) or a single domain antibody, for example a VH domain) or antibody mimetic protein.

[0599] In one embodiment, the treatment is used in combination with an existing therapy or therapeutic agent, for example an existing anti-cancer therapy. Thus, in another aspect, the invention also relates to a combination therapy comprising administration of an activatable binding molecule or pharmaceutical composition of the invention and an anti-cancer therapy. The anti-cancer therapy may include a therapeutic agent or radiation therapy and includes gene therapy, viral therapy, RNA therapy bone marrow transplantation, nanotherapy, targeted anti-cancer therapies or oncolytic drugs. Examples of other therapeutic agents include other checkpoint inhibitors, antineoplastic agents, immunogenic agents, attenuated cancerous cells, tumor antigens, antigen presenting cells such as dendritic cells pulsed with tumor-derived antigen or nucleic acids, immune stimulating cytokines (e.g., IL-2, IFNa2, GM-CSF), targeted small molecules and biological molecules (such as components of signal transduction pathways, e.g. modulators of tyrosine kinases and inhibitors of receptor tyrosine kinases, and agents that bind to tumorspecific antigens, including EGFR antagonists), an anti-inflammatory agent, a cytotoxic agent, a radiotoxic agent, or an immunosuppressive agent and cells transfected with a gene encoding an immune stimulating cytokine (e.g., GM-CSF), chemotherapy. In one embodiment, the activatable binding molecule is used in combination with surgery.

[0600] In a specific embodiment of the present invention, the activatable binding molecule is administered concurrently with a chemotherapeutic agent or with radiation therapy. In another specific embodiment, the chemotherapeutic agent or radiation therapy is administered prior or subsequent to administration of the composition of the present invention, preferably at least an hour, five hours, 12 hours, a day, a week, a month, more preferably several months (e. g. up to three months), prior or subsequent to administration of composition of the present invention.

[0601] In some embodiments, the activatable binding molecule or pharmaceutical composition of the invention may be administered with two or more therapeutic agents.

[0602] The activatable binding molecule or pharmaceutical composition of the invention may be administered at the same time or at a different time as the other therapy or therapeutic compound or therapy, e.g., simultaneously, separately or sequentially.

[0603] In another aspect, the invention relates to an activatable binding molecule or pharmaceutical composition of the invention for use in the treatment or prevention of inflammatory disease.

[0604] In another aspect, the invention relates to the use of an activatable binding molecule or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment or prevention of inflammatory disease.

[0605] In another aspect, the invention relates to a method for treating an inflammatory disease comprising administering a therapeutically effective amount of an activatable binding molecule as described herein, or a pharmaceutical composition as described herein.

[0606] As will be appreciated by the skilled person the term “inflammatory disease” refers to a disease in which tissues are inflamed, with, for example, increased recruitment of white blood cells to a tissue, which may cause swelling, pain and loss of function. Inflammatory diseases include, for example, arthritis, asthma, Crohn’s disease, colitis, dermatitis, irritable bowel syndrome. Notably, chronic inflammation is often a precursor to cancer. Inflammatory diseases also include autoimmune diseases and other inflammatory disorders such as myositis, ankylosing spondylitis, vasculitis.

[0607] In embodiments, the inflammatory disease is an inflammatory disease that expresses abnormally high levels of LGR5, for example inflammatory disease which overexpress LGR5. The term “overexpress” as used herein means that the cells express more LGR5 than that observed in normal cells not suffering from inflammatory disease. The cells may express 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% more LGR5 than that observed in normal cells. The skilled person will be well aware how to assess whether an inflammatory disease overexpresses LGR5. Methods include for example, using fluorescence in-situ hybridization, immunohistochemistry approaches, flow cytometry, RT-PCR.

[0608] Without wishing to be bound by theory, the inventors hypothesize that chronically inflamed tissues result in the upregulation of stem cell pathways and overexpression of LGR5. By targeting LGR5, using the activatable binding molecule of the present invention, the inventors can therefore target cells affected by inflammatory disease.

[0609] The present inventors have found that by utilising the activatable binding molecule of the present invention, a toxic payload, in the form of an antibody drug conjugate (ADC) can be delivered to LGR5-expressing cells to kill those cells without systemic toxicity.

[0610] In another aspect, the invention provides a kit comprising an activatable binding molecule or a pharmaceutical composition as described herein.

[0611] In another aspect, the invention provides a kit for the treatment or prevention of a disease prognosis or monitoring disease comprising an activatable binding molecule of the invention. Such a kit may contain other components, packaging, instructions. The kit may include a labeled activatable binding molecule of the invention as described above and one or more compounds for detecting the label.

[0612] The invention in another aspect provides an antibody of the invention packaged in lyophilized form or packaged in an aqueous medium.

[0613] Methods

[0614] The present invention also relates to methods of producing an activatable binding molecule. A method of preparing an activatable binding molecule comprising: conjugating a shield moiety comprising MM-CM-EX-LM- to an antibody or fragment thereof (AB), to form an activatable binding molecule,

[0615] wherein the shield moiety is conjugated to AB via the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

[0616] In an embodiment the method comprises conjugating the shield moiety via the side chain of an amino acid and / or a glycan present in the CH1 domain, CL domain, hinge domain and / or Fc domain of AB. In an embodiment the method comprises conjugating the shield moiety via the side chain of a naturally occurring amino acid, a genetically engineered amino acid or a genetically engineered unnatural amino acid. In an embodiment the method comprises conjugating the shield moiety via one or more thiol groups of cysteine residues.

[0617] In an embodiment the method comprises modifying one or more interchain disulfide bond of said antibody or fragment thereof, prior to conjugating the shield moiety. Modifying the one or more interchain disulfide bond may comprise reducing one or more interchain disulfide bond. Reducing one or more interchain disulfide bond may be performed using any suitable reducing agent, for example TCEP, DTT etc. In an embodiment LM is conjugated to at least one thiol produced from reducing one or more interchain disulfide bond. As such the method may comprise:

[0618] reducing one or more interchain disulfide bond present in an antibody or fragment thereof to produce one or more thiol group;

[0619] conjugating at least one shield moiety comprising MM-CM-EX-LM- to said one or more thiol group, to form an activatable binding molecule.

[0620] The present invention also relates to methods of screening masking moieties (MM) for use in activatable binding molecules. An aspect of the invention relates to a method of screening masking moieties (MM) comprising:

[0621] preparing one or more variant MM;

[0622] preparing a library of one or more shield moieties to be screened, wherein a shield moiety comprises MM-CM-CS1;

[0623] preparing an antibody or fragment comprising AB-LM-CS2;

[0624] conjugating AB-LM-CS2 to one or more of MM-CM-CS1; and

[0625] determining the reduction or inhibition on AB binding elicited by said shield moiety;

[0626] wherein MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;

[0627] CM represent a cleavable moiety;

[0628] LM represents a linking moiety;

[0629] CS1 represents one part of a conjugation system; CS2 represents the second complimentary part of said conjugation system; and AB represents an antibody or antigen binding fragment thereof.

[0630] The conjugation system CS1 CS2 may be any catcher tag system described herein. The conjugation system may comprise a click pair as described herein. CS1 and CS2 indicate that they are the complimentary parts of a conjugation system that once brought into proximity link together.

[0631] The CM may be any cleavable moiety as described herein. The LM may be any linking moiety as described herein. The LM is conjugated to one part of the conjugation system. For example, where the conjugation system comprises a click reaction the LM may be conjugated to one part of the click pair. A click pair generally refers to i) a ketone or aldehyde and an alkoxyamine (e.g. hydroxylamine) or hydrazine; ii) an azide and an alkyne; iii) an amine and an acyl halide or carboxylic acid; iv) electron-rich dienophile (e.g. a 1,3-nitrone alkene) and an electron-poor diene (e.g. tetrazine); and iii) a strained alkene or alkyne (e.g. norbornene or cyclooctyne) and a tetrazine. The CM is then conjugated to the complimentary part of the click pair.

[0632] In embodiments where a catcher tag conjugation system is used the LM may be conjugated to the catcher or the tag part of the conjugation system, the CM is the conjugated to the complementary catcher or tag as appropriate.

[0633] Preparing a library of shield moieties may comprise developing a series of amino acid sequences or glycan sequences with modulated binding affinity for AB. The amino acid sequences or glycan sequences of the library may also be developed to interfere with AB binding via steric hindrance. In certain embodiments the library of MMs may be based on a natural binding partner of AB, for example the library of MMs may comprise an epitope of the AB. The library of MMs may comprise an epitope with one or more amino acid substitutions compared to the wild-type sequence of said epitope. In some embodiments the library of MM comprises amino acid sequences with high homology or similarity to a natural binding partner of the AB. In some embodiments the library of MM comprises amino acid sequences with more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% homology or similarity to any natural binding partner of the AB. In some embodiments the library of MM comprises amino acid sequences with more than 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any natural binding partner of the AB.

[0634] In some embodiments, the library of MMs may be based on a non-natural binding partner of AB, i.e. the library of MMs has been selected to be substantially different to a natural binding partner of the AB. In an embodiment the library of MMs contains no or substantially no homology to any natural binding partner of the AB. In some embodiments the library of MMs comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% homology or similarity to any natural binding partner of the AB. In some embodiments, the library of MMs comprises no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% identity to any natural binding partner of the AB.

[0635] The MM may comprise an amino acid sequence. The amino acid sequence may have a length of up to 15 amino acids, a length of up to 20 amino acids, a length of up to 25 amino acids, a length of up to 30 amino acids, a length of up to 35 amino acids, a length of up to 40 amino acids, a length of up to 45 amino acids, a length of up to 50 amino acids, a length of up to 60 amino acids, a length in the range of 10-60 amino acids, a length in the range of 15-60 amino acids, a length in the range of 20-60 amino acids, a length in the range of 25-60 amino acids, a length in the range of 30-60 amino acids, a length in the range of 35-60 amino acids, a length in the range of 40-50 amino acids, a length in the range of 45-60 amino acids, a length in the range of 10-40 amino acids, a length in the range of 15-40 amino acids, a length in the range of 20-40 amino acids, a length in the range of 25-40 amino acids, a length in the range of 30-40 amino acids, a length in the range of 35-40 amino acids, a length in the range of 10-30 amino acids, a length in the range of 15-30 amino acids, a length in the range of 20-30 amino acids, a length in the range of 25-30 amino acids, a length in the range of 10-20 amino acids, a length in the range of 10-15 amino acids, or a length of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0636] Unless otherwise defined herein, scientific and technical terms used in connection with the present disclosure shall have the meanings that are commonly understood by those of ordinary skill in the art. While the foregoing disclosure provides a general description of the subject matter encompassed within the scope of the present invention, including methods, as well as the best mode thereof, of making and using this invention, the following examples are provided to further enable those skilled in the art to practice this invention and to provide a complete written description thereof. However, those skilled in the art will appreciate that the specifics of these examples should not be read as limiting on the invention, the scope of which should be apprehended from the claims and equivalents thereof appended to this disclosure. Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

[0637] All documents mentioned in this specification are incorporated herein by reference in their entirety, including references to gene accession numbers and references to patent publications.

[0638] "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example " A and / or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described.

[0639] The invention is further described in the non-limiting examples.

[0640] Numbered Embodiments

[0641] 1. An activatable binding molecule, comprising the structure of formula (I):

[0642] MM-CM-EX-LM-AB (I)

[0643] wherein AB represents an antibody or antigen binding fragment thereof capable of binding a target when the binding molecule is in an activated state;

[0644] MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;

[0645] CM represent a cleavable moiety;

[0646] EX represents an extension moiety;

[0647] LM represents a linking moiety wherein the linking moiety is covalently attached via one or more amino acid side chain present in the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

[0648] 2. The activatable binding molecule according to embodiment 1, wherein the LM is attached via one or more amino acid side chain and / or glycan, present in the CH1 domain, CL domain, hinge domain and / or Fc domain of AB, optionally wherein the amino acid side chain comprises the side chain of a naturally occurring amino acid, a genetically engineered amino acid or a genetically engineered unnatural amino acid.

[0649] 3. The activatable binding molecule according to embodiment 1 or 2, wherein the linking moiety, LM, is attached via one or more thiol groups of cysteine residues.

[0650] 4. The activatable binding molecule according to any one of embodiments 1 to 3, wherein the LM, comprises and interchain linker.

[0651] 5. The activatable binding molecule according to any one of embodiments 1 to 4, wherein LM, is attached to AB at one or more interchain disulfide position.

[0652] 6. The activatable binding molecule according to any one of embodiments 1 to 5, wherein the linking moiety, LM, comprises and interchain disulphide re-bridging linker.

[0653] 7. The activatable binding molecule according to any one of embodiments 1 to 6, wherein LM comprises between 1 and 4 re-bridging moieties comprising the structure of formula (II)

[0654]

[0655] wherein:

[0656] ZAis a functional linking group;

[0657] Pep indicates the position where the re-bridging moiety is attached to the AB, such that the re-bridging moiety re-bridges a reduced interchain disulfide bond in the AB

[0658]

[0659] indicates the position where the re-bridging moiety is attached to a further moiety.

[0660] 8. The activatable binding molecule according to any one of embodiments 1 to 7, wherein LM comprises between 1 and 4 re-bridging moieties comprising the structure of formula (Ila):

[0661] AL. A

[0662]

[0663] wherein:

[0664] one or two of A1, A2, and A3are N and the other one or two of A1, A2, and A3are CH, or all three of A1, A2, and A3are N or C;

[0665] a and b are integers selected from 0 or 1;

[0666] X is selected from N, NRN, O and S, wherein RNis H or C1-2 alkyl;

[0667] Pep indicates where the re-bridging moiety is attached to AB.

[0668] 9. The activatable binding molecule according to any one of embodiments 1 to 8, wherein LM comprises between 1 and 4 re-bridging moieties comprising the structure of formula (Illa):

[0669]

[0670] wherein two of A1, A2and A3are N and the other of A1, A2and A3is CH;

[0671] X is selected from N, O and S;

[0672] Pep indicates where the re-bridging moiety is attached to AB.

[0673] 10. The activatable binding molecule according to any one of embodiments 8 to 9, wherein A1and A2are N.

[0674] 11. The activatable binding molecule according to any one of embodiments 8 to 10, wherein A1and A3are N.

[0675] 12. The activatable binding molecule according to any one of embodiments 8 to 11, wherein X is selected from NRN, O and S, where RNis H or C1-2 alkyl, and there is an extension moiety, EX, attached to X via a linker.

[0676] 13. The activatable binding molecule according to any one of embodiments 8 to 12, wherein the LM comprises the moiety of formula (lllb):

[0677] wherein Q1is:

[0678]

[0679] where a1 = 0 to 5, b1 = 0 to 16, d = 0 to 5, d1 is 0 to 16, and b1 + d1 = 0 to 16;

[0680] and YLis a functional linking moiety. 14. The activatable binding molecule according to embodiment 13, wherein YLis selected from the group consisting of:

[0681] (

[0682]

[0683] b)

[0684] 15. The activatable binding molecule according to any one of embodiments 8 to 14, wherein the LM comprises the moiety of formula (lllc):

[0685] where Q2 is:

[0686]

[0687] where a2 = 0 to 5, b2 = 0 to 16, c2 = 0 to 5, d2 is 0 to 16, and b2 + d2 = 0 to 16.

[0688] 16. The activatable binding molecule according to any one of embodiments 8 to 15, wherein the LM comprises the moiety of formula (IIId): Where Q3is:

[0689]

[0690] wherein Q4is a single bond, or O, where Qxis such that Q4is an amino-acid residue, a dipeptide residue or a tripeptide residue, and L is a group for attachment to the extension moiety, EX.

[0691] 17. The activatable binding molecule according to embodiment 16, wherein L is selected from:

[0692] (a) a single bond;

[0693] (b) -C(=0)-;

[0694] (c) -NH-; and

[0695]

[0696] 18. The activatable binding molecule according to any one of embodiments 8 to 17, wherein the LM is of formula (Hie):

[0697]

[0698] where D is the extension moiety EX.

[0699] 19. The activatable binding molecule according to any one of embodiments 1 to 18, wherein the LM or AB is conjugated to further moiety.

[0700] 20. The activatable binding molecule according to embodiment 19, wherein the further moiety is a labelling moiety.

[0701] 21. The activatable binding molecule according to embodiment 20, wherein the labelling moiety is a fluorophore, a fluorescer, a radiolabel, a chemiluminescer, a nuclear magnetic resonance active label, a photosensitizer or a biotin tag.

[0702] 22. The activatable binding molecule according to embodiment 19, wherein the further moiety is a cell killing agent, an immune-modulating payload, a macrophage class switching agent, antisense oligonucleotide or a light activatable payload.

[0703] 23. The activatable binding molecule according to embodiment 22, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

[0704] 24. The activatable binding molecule according to embodiment 22, wherein the cell killing agent comprises a cytotoxin.

[0705] 25. The activatable binding molecule according to embodiment 22, wherein said cytotoxin is selected from:

[0706] i) a peptide toxin; or

[0707] ii) a chemical toxin.

[0708] 26. The activatable binding molecule according to embodiment 24 or 25, wherein the cytoxin is selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines, camptothecin analogues, topoisomerase inhibitors and doxorubicin. 27. The activatable binding molecule according to any one of embodiments 8 to 26, wherein X is N and there is one active agent and one extension moiety EX, each attached to X via a linker.

[0709] 28. The activatable binding molecule according to embodiment 27, wherein the LM comprises the moiety of formula (lllf):

[0710]

[0711] wherein Q1and YLare as defined in either embodiments 13 or 14;

[0712] Q5is:

[0713]

[0714] where a5 = 0 to 5, b5 = 0 to 16, c5 = 0 to 5, d5 is 0 to 16, and b5 + d5 = 0 to 16;

[0715] and YL2is a functional linking moiety.

[0716] 29. The activatable binding molecule according to embodiment 28, wherein YL2is selected from the group consisting of:

[0717] N""" N

[0718] (

[0719]

[0720] b) -C(=0)NH-

[0721] 30. The activatable binding molecule according to any one of embodiment 27 to 29, wherein the LM comprises the moiety of formula (Illg):

[0722]

[0723] wherein Q2is as defined in embodiment 15;

[0724] Q6is:

[0725] where a6 = 0 to 5, b6 = 0 to 16, c6 = 0 to 5, d6 is 0 to 16, and b6 + d6 = 0 to 16.

[0726] 31. The activatable binding molecule according to any one of embodiment 27 to 30, wherein the LM comprises the moiety of formula (lllh):

[0727]

[0728] wherein Q3is as defined in either embodiment 16 or 17;

[0729] Q7is:

[0730]

[0731] / .-A X

[0732] ? A -A-'- Ar

[0733] H

[0734] wherein Q8is a single bond, or

[0735] , where Qxis such that Q4is an amino-acid residue, a dipeptide residue or a tripeptide residue, and L2is a group for attachment to an active agent or EX.

[0736] 32. The activatable binding molecule according to embodiment 31, wherein L2is selected from:

[0737] (a) a single bond;

[0738] (b) -C(=0)-;

[0739] (c) -NH-; and

[0740]

[0741] (d)

[0742] 33. The activatable binding molecule according to any one of embodiment 27 to 32, wherein the LM is of formula (Illi):

[0743]

[0744] where D is the active agent and D2 is the extension moiety EX.

[0745] 34. The activatable binding molecule according to any one of embodiments 7 to 33, wherein the LM comprises Formula (IVA), shown below: Z1

[0746] Q1

[0747] Z1- Q1- Q2- Q3- (W1)r- (W2)s- Q3- Q2- Q1- Z1

[0748] Q1

[0749]

[0750] (Formula IVA)

[0751] wherein:

[0752] Z1is a re-bridging moiety of Formula (II) as defined in any one of embodiments 7 to 9 above that is attached to an antibody at the positions shown in Formula (II);

[0753] each Q1is independently a bond or a group of Formula Va:

[0754]

[0755] Formula IVa

[0756] wherein:

[0757] Q1Ais absent or selected from a C(=O), OC(=O), NR7C(=O), C(=NR8) and C(=S);

[0758] Q1Bis selected from O, N, S and CR9CR10;

[0759] R7, R8, R9and R10are independently selected from hydrogen and C1-C4alkyl;

[0760] R20and R21are independently selected from a bond, a C1-C10 alkylene and a C1-C10 alkylene containing O in the backbone; and v is an integer selected from 0, 1 or 2;

[0761] each Q2is independently selected from N and CR11, wherein R11is selected from hydrogen and C1-C4alkyl;

[0762] each Q3is independently a group of Formula Vb:

[0763]

[0764] Formula Vb

[0765] wherein:

[0766] Q3Ais selected from NR12, O and S;

[0767] Q3Band Q3Care independently selected from a bond, O and NR13; R12and R13are independently selected from hydrogen and C1-C4alkyl; R22and R23are independently selected from a bond, a C1-C10alkylene and a C1-C10alkylene containing O in the backbone;

[0768] Q3Dis absent or selected from C(=O), OC(=O), NR12C(=O) and C(=O)NR12;

[0769] W1is a group of Formula Vc or Formula Ve:

[0770]

[0771]

[0772] Formula Vc Formula Ve wherein:

[0773] W1Ais selected from N and CR14, wherein R14is a selected from hydrogen and C1-C4alkyl;

[0774] Lwis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing amino, O or N in the backbone;

[0775] Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl or a 6-membered cycloalkyl, wherein Xi is selected from CH or N;

[0776] W1Bis a group selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - OC(=O)O-, -NR15C(=O)-, -C(=O)NR15-, -NR15C(=O)NR16-, -C(=NR15)- and -C(=S)-;

[0777] R15and R16are independently selected from hydrogen and C1-C4alkyl; R23and R24are independently selected from a bond, Ci-Cwalkylene and Ci-Cwalkylene containing O in the backbone;

[0778] m is an integer selected from 1 or 2;

[0779] W1Cis selected from Formula W01or Formula W02: W02

[0780] FG' W01

[0781]

[0782] Formula W01Formula W02

[0783] wherein:

[0784] W01is a group of Formula W03:

[0785] QW2

[0786]

[0787] Formula W03

[0788] wherein:

[0789] QW1is absent or selected from a C1-C12alkylene and a C1-C12alkylene containing O or N in the backbone; and

[0790] QW2is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - NHC(=O)-, and -C(=O)NH-;

[0791] y is an integer selected from 0 or 1;

[0792] XW1is selected from O or NH;

[0793] XW2is selected from hydrogen, C1-C4alkyl, ORx1and NRx1Rx2, wherein Rx1and Rx2are independently selected from hydrogen and C1-C4alkyl;

[0794] WQ2

[0795]

[0796] indicates the position where XW1is attached to a group of formula W02;

[0797] R24

[0798]

[0799] indicates the position where W01is attached to a group of formula R24

[0800] W02is a group of Formula WC4: QW1A

[0801]

[0802] Formula WC4

[0803] wherein:

[0804] QW1Ais absent or selected from a C1-C12alkylene and a C1-C12alkylene containing O or N in the backbone; and

[0805] QW2Ais selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - NHC(=O)-, and -C(=O)NH-; LQ1is a linker comprising one or more groups selected from a C1-C20alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereof;

[0806] XW3is selected from O or NH;

[0807] w

[0808]

[0809] indicates the position where XW4is attached to another group of formula W02via the QW2Asubstituent group, or

[0810] W

[0811]

[0812] indicates the position where XW4is attached to one of the following groups: a hydrogen or - C(=O)RX3, wherein Rx3is selected from hydrogen and C1-C4alkyl; W31

[0813]

[0814] indicates the position where

[0815]

[0816] is attached to the group of formula W01via the XW1group; XW4is selected from O or NH;

[0817] LQ1is a linker comprising one or more groups selected from a C₁-C₂₀alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereof t is an integer selected from 1 to 8;

[0818] FG’ is a functional linking moiety;

[0819] L1is a linker; and

[0820] D is a further moiety selected from a shield arm or active agent;

[0821] W2is a group of Formula Vd:

[0822] R27

[0823] V\PC

[0824] R25

[0825]

[0826] Formula IVd

[0827] wherein:

[0828] W2Ais selected from N and CR17;

[0829] W2Bis a group selected from N, CR18, -C(=O)N- and -C(=O)CR18-; W2Cis a group selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR17C(=O)-, -C(=O)NR17-, -NR17C(=O)NR18-, -C(=NR17)- and -C(=S)-;

[0830] L2Wis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing amino, O or N in the backbone;

[0831] R17and R18are independently selected from hydrogen and C1-C4alkyl; R25and R26are independently selected from a bond, C₁-C₆alkylene and C₁-C₆alkylene containing O in the backbone; and

[0832] FG’, L1and D are as defined above;

[0833] r is an integer selected from 0 to 12;

[0834] s is an integer selected from 0 to 6; and

[0835] wherein r and s are selected such that the total number of active agents per conjugate is between 1 and 12.

[0836] 35. The activatable binding molecule according to any one of embodiments 1 to 34, wherein the antigen binding fragment is selected from a scFv, Fab, Fab’or F(ab’)2.

[0837] 36. The activatable binding molecule according to any one of embodiments 1 to 35, wherein the masking moiety, MM, comprises an amino acid sequence that binds to AB and inhibits the interaction of AB with the target.

[0838] 37. The activatable binding molecule according to embodiment 36, wherein the amino acid sequence that binds comprises an epitope which binds to AB, optionally wherein the epitope comprises one or more amino acid substitution which modulates the affinity of the epitope to AB.

[0839] 38. The activatable binding molecule according to any one of embodiments 1 to 35, wherein the MM comprises an amino acid sequence that sterically hinders the interaction of AB with the target.

[0840] 39. The activatable binding molecule according to any one of embodiments 1 to 38, wherein the cleavable moiety, CM, is enzymatically cleavable or acid labile.

[0841] 40. The activatable binding molecule according to embodiment 39, wherein the CM is protease cleavable.

[0842] 41. The activatable binding molecule according to embodiment 40, wherein the protease is selected from a metalloprotease, a threonine protease, a serine protease, an aspartic acid protease, or a cysteine protease

[0843] 42. The activatable binding molecule according to embodiment 39 or 40, wherein the protease is selected from a matrix metalloproteinase (MMP), cathepsin, a kallikrein, a disintegrin and metalloproteinase (ADAM), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMts), Granzyme B.

[0844] 43. The activatable binding molecule according to embodiment 42, wherein the MMP is selected from MMP-1, -3, -7, -8, -10, -12, -13, -20, -27, -2, -9, -11, -17, -25, -14, -15, -16, -24,-19, -21, -23, -26, and / or -28. 44. The activatable binding molecule according to any one of embodiments 1 to 43, wherein the extension moiety, EM, comprises a PEG linker, polysarcosine linker and / or a Gly / Ser linker second linking moiety.

[0845] 45. The activatable binding molecule according to any one of embodiments 1 to 44, wherein the EM comprises a conjugation system, which allows for conjugation of MM-CM to LM-AB.

[0846] 46. The activatable binding molecule according to embodiment 45, wherein the conjugation system comprises a catcher-tag system.

[0847] 47. The activatable binding molecule according to embodiment 46, wherein the catcher-tag system is selected from SpyCatcher-SpyTag system, SnoopTag-SnoopCatcher system, Spy-Stapler-SpyTag system, Spyligase-SpyTag system, SdyCatcher-SpyTag system, SilkCatcher-SilkTag, N1 / 2-C1 / 2 system, N4 / 5-C4 / 5 system, N5 / 6-C5 / 6 system, N6 / 7b-C6 / 7b system, CnaB2Ctacher-CnaB2Tag system, PilinC-lsopeptagC system, PilinN-lsopeptagN system, DogCatcher-DogTag system, 4oq1c- 4oq1Tsystem, 3kptCc- 3kptCT, Snoopligase- SnoopTagJr, Jo-In system.

[0848] 48. The activatable binding molecule according to embodiment 45, wherein click chemistry is used to conjugate MM-CM to LM-AB.

[0849] 49. The activatable binding molecule according to embodiment 48, wherein the click chemistry is used to conjugate together MM-CM to LM-AB is selected from an inverse electron demand Diels-Alder (iEDDA), a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and / or a strained-promoted azide-alkyne click chemistry (SPAAC) reaction.

[0850] 50. The activatable binding molecule according to any one of embodiments 1 to 49, wherein AB binds to LRG5.

[0851] 51. The activatable binding molecule according to any one of embodiment 1 to 50, wherein AB comprises and antibody or antigen binding fragment thereof that binds to LRG5.

[0852] 52. The activatable binding molecule according to any one of embodiment 50 or 51, wherein AB binds an epitope located within amino acids 22-37 of SEQ ID NO.1.

[0853] 53. The activatable binding molecule according to any one of embodiment 50 to 52, wherein AB binds an epitope which consists of amino acids 22-37 of SEQ ID NO.1.

[0854] 54. The activatable binding molecule according to any one of embodiment 50 to 53, wherein the VH of the antibody comprises the following CDR1, CDR2 and CDR3:

[0855] a) a CDR1 of SEQ ID No. 50 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 51 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 52 or a sequence with at least 90% homology thereto; or

[0856] b) a CDR1 of SEQ ID No. 2 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 3 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 4 or a sequence with at least 90% homology thereto; or

[0857] c) a CDR1 of SEQ ID No. 8 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 9 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 10 or a sequence with at least 90% homology thereto; or d) a CDR1 of SEQ ID No. 14 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 15 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 16 or a sequence with at least 90% homology thereto; or

[0858] e) a CDR1 of SEQ ID No. 20 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 21 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 22 or a sequence with at least 90% homology thereto; or

[0859] f) a CDR1 of SEQ ID No. 26 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 27 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 28 or a sequence with at least 90% homology thereto; or

[0860] g) a CDR1 of SEQ ID No. 32 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 33 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 34 or a sequence with at least 90% homology thereto; or

[0861] h) a CDR1 of SEQ ID No. 38 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 39 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 40 or a sequence with at least 90% homology thereto; or

[0862] i) a CDR1 of SEQ ID No. 44 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 45 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 46 or a sequence with at least 90% homology thereto; or

[0863] j) a CDR1 of SEQ ID No. 56 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 57 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 58 or a sequence with at least 90% homology thereto; or

[0864] k) a CDR1 of SEQ ID No. 62 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 63 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 64 or a sequence with at least 90% homology thereto; or

[0865] l) a CDR1 of SEQ ID No. 68 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 69 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 70 or a sequence with at least 90% homology thereto; or

[0866] m) a CDR1 of SEQ ID No. 74 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 75 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 76 or a sequence with at least 90% homology thereto.

[0867] 55. An activatable binding molecule according to any one of embodiment 50 to 54, wherein the VL of the antibody comprises the following CDR1, CDR2 and CDR3:

[0868] a) a CDR1 of SEQ ID No. 53 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 54 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 55 or a sequence with at least 90% homology thereto; or

[0869] b) a CDR1 of SEQ ID No. 5 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 6 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 7 or a sequence with at least 90% homology thereto; or c) a CDR1 of SEQ ID No. 11 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 12 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 13 or a sequence with at least 90% homology thereto; or

[0870] d) a CDR1 of SEQ ID No. 17 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 18 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 19 or a sequence with at least 90% homology thereto; or

[0871] e) a CDR1 of SEQ ID No. 23 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 24 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 25 or a sequence with at least 90% homology thereto; or

[0872] f) a CDR1 of SEQ ID No. 29 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 30 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 31 or a sequence with at least 90% homology thereto; or

[0873] g) a CDR1 of SEQ ID No. 35 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 36 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 37 or a sequence with at least 90% homology thereto; or

[0874] h) a CDR1 of SEQ ID No. 41 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 42 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 43 or a sequence with at least 90% homology thereto; or

[0875] i) a CDR1 of SEQ ID No. 47 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 48 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 49 or a sequence with at least 90% homology thereto; or

[0876] j) a CDR1 of SEQ ID No. 59 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 60 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 61 or a sequence with at least 90% homology thereto; or

[0877] k) a CDR1 of SEQ ID No. 65 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 66 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 67 or a sequence with at least 90% homology thereto; or

[0878] l) a CDR1 of SEQ ID No. 71 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 72 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 73 or a sequence with at least 90% homology thereto; or

[0879] m) a CDR1 of SEQ ID No. 77 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 78 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 79 or a sequence with at least 90% homology thereto.

[0880] 56. The activatable binding molecule according to any one of embodiment 50 to 55, comprising a VH sequence selected from SEQ ID NOs: 96, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104.

[0881] 57. The activatable binding molecule according to any one of embodiment 50 to 56, comprising a VL sequence selected from SEQ ID NOs: 97, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105.

[0882] 58. The antibody or fragment thereof according to any one of embodiment 50 to 57 comprising: i) a VH sequence of SEQ ID NO: 96 and a VL sequence of SEQ ID NO: 97; b) a VH sequence of SEQ ID NO: 80 and a VL sequence of SEQ ID NO: 81;

[0883] c) a VH sequence of SEQ ID NO: 82 and a VL sequence of SEQ ID NO: 83;

[0884] d) a VH sequence of SEQ ID NO: 84 and a VL sequence of SEQ ID NO: 85;

[0885] e) a VH sequence of SEQ ID NO: 86 and a VL sequence of SEQ ID NO: 87;

[0886] f) a VH sequence of SEQ ID NO: 88 and a VL sequence of SEQ ID NO: 89;

[0887] g) a VH sequence of SEQ ID NO: 90 and a VL sequence of SEQ ID NO: 91;

[0888] h) a VH sequence of SEQ ID NO: 92 and a VL sequence of SEQ ID NO: 93;

[0889] i) a VH sequence of SEQ ID NO: 94 and a VL sequence of SEQ ID NO: 95;

[0890] j) a VH sequence of SEQ ID NO: 98 and a VL sequence of SEQ ID NO: 99;

[0891] k) a VH sequence of SEQ ID NO: 100 and a VL sequence of SEQ ID NO: 101

[0892] l) a VH sequence of SEQ ID NO: 102 and a VL sequence of SEQ ID NO: 103; or

[0893] m) a VH sequence of SEQ ID NO: 104 and a VL sequence of SEQ ID NO: 105.

[0894] 59. The activatable binding molecule according to any one of embodiment 50 to 58, comprising a sequence of an antibody clone as shown in Table 3 or a sequence with at least 70%, 80% or 90% homology thereto.

[0895] 60. The activatable binding molecule according to embodiment 50 or 59 wherein the fragment is an scFV comprising SEQ ID NO. 211 or a sequence with at least 70%, 80% or 90% homology thereto.

[0896] 61. The activatable binding molecule according to any one of embodiment 50 to 53, wherein AB comprises and antibody or antigen binding fragment thereof that binds to LRG5, wherein the VH of the antibody or antigen binding fragment comprises the following CDR1, CDR2 and CDR3: a) a CDR1 of SEQ ID No. 50 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 51 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 52 or a sequence with at least 90% homology thereto; or

[0897] b) a CDR1 of SEQ ID No. 2 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 3 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 4 or a sequence with at least 90% homology thereto; or

[0898] c) a CDR1 of SEQ ID No. 8 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 9 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 10 or a sequence with at least 90% homology thereto; or

[0899] d) a CDR1 of SEQ ID No. 14 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 15 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 16 or a sequence with at least 90% homology thereto; or

[0900] e) a CDR1 of SEQ ID No. 20 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 21 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 22 or a sequence with at least 90% homology thereto; or

[0901] f) a CDR1 of SEQ ID No. 26 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 27 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 28 or a sequence with at least 90% homology thereto; or g) a CDR1 of SEQ ID No. 32 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 33 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 34 or a sequence with at least 90% homology thereto; or

[0902] h) a CDR1 of SEQ ID No. 38 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 39 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 40 or a sequence with at least 90% homology thereto; or

[0903] i) a CDR1 of SEQ ID No. 44 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 45 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 46 or a sequence with at least 90% homology thereto; or

[0904] j) a CDR1 of SEQ ID No. 56 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 57 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 58 or a sequence with at least 90% homology thereto; or

[0905] k) a CDR1 of SEQ ID No. 62 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 63 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 64 or a sequence with at least 90% homology thereto; or

[0906] l) a CDR1 of SEQ ID No. 68 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 69 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 70 or a sequence with at least 90% homology thereto; or

[0907] m) a CDR1 of SEQ ID No. 74 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 75 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 76 or a sequence with at least 90% homology thereto.

[0908] 62. The activatable binding molecule according to embodiment 61 which binds to LGR5 wherein the VL of the antibody comprises the following CDR1, CDR2 and CDR3:

[0909] a) a CDR1 of SEQ ID No. 53 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 54 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 55 or a sequence with at least 90% homology thereto; or

[0910] b) a CDR1 of SEQ ID No. 5 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 6 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 7 or a sequence with at least 90% homology thereto; or

[0911] c) a CDR1 of SEQ ID No. 11 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 12 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 13 or a sequence with at least 90% homology thereto; or

[0912] d) a CDR1 of SEQ ID No. 17 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 18 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 19 or a sequence with at least 90% homology thereto; or

[0913] e) a CDR1 of SEQ ID No. 23 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 24 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 25 or a sequence with at least 90% homology thereto; or f) a CDR1 of SEQ ID No. 29 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 30 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 31 or a sequence with at least 90% homology thereto; or

[0914] g) a CDR1 of SEQ ID No. 35 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 36 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 37 or a sequence with at least 90% homology thereto; or

[0915] h) a CDR1 of SEQ ID No. 41 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 42 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 43 or a sequence with at least 90% homology thereto; or

[0916] i) a CDR1 of SEQ ID No. 47 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 48 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 49 or a sequence with at least 90% homology thereto; or

[0917] j) a CDR1 of SEQ ID No. 59 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 60 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 61 or a sequence with at least 90% homology thereto; or

[0918] k) a CDR1 of SEQ ID No. 65 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 66 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 67 or a sequence with at least 90% homology thereto; or

[0919] l) a CDR1 of SEQ ID No. 71 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 72 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 73 or a sequence with at least 90% homology thereto; or

[0920] m) a CDR1 of SEQ ID No. 77 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 78 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 79 or a sequence with at least 90% homology thereto.

[0921] 63. The activatable binding molecule according to embodiment 61 or 62, comprising a VH sequence selected from SEQ ID NOs: 96, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104. 64. The activatable binding molecule according to embodiment 61 to 63, comprising a VL sequence selected from SEQ ID NOs: 97, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105. 65. A pharmaceutical composition comprising an activatable binding molecule according to any one of embodiments 1 to 64 and a pharmaceutical carrier.

[0922] 66. An activatable binding molecule according to any one of embodiments 1 to 64, or a pharmaceutical composition according to embodiment 65 for use as a medicament.

[0923] 67. An activatable binding molecule according to any one of embodiment 1 to 64, or a pharmaceutical composition according to embodiment 65 for use in the treatment or diagnosis of a cancer or an inflammatory disease.

[0924] 68. A method for treating a cancer comprising administering a therapeutically effective amount of an activatable binding molecule according to any one of embodiment 1 to 64, or a pharmaceutical composition according to embodiment 65. 69. The activatable binding molecule for use of embodiment 67 or the method of embodiment 68, wherein the cancer is an LGR5-positive cancer.

[0925] 70. The activatable binding molecule for use of embodiment 69 or the method of embodiment 69 wherein the cancer overexpresses LGR5.

[0926] 71. The activatable binding molecule for use of any of embodiment 67, 69 or 70, or the method of any of embodiment 68 to 70 wherein the cancer is selected from cancer of the head or neck, uterine cancer, colorectal cancer, stomach cancer, carcinoma of the endometrium, cancer of the esophagus, leukemia, such as acute lymphoblastic leukemia (ALL), liver cancer, such as hepatocellular carcinoma, or pancreatic cancer.

[0927] 72. A method of preparing an activatable binding molecule comprising:

[0928] conjugating a shield moiety comprising MM-CM-EX-LM- to an antibody or fragment thereof (AB), to form an activatable binding molecule,

[0929] wherein the shield moiety is conjugated to AB via the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

[0930] 73. The method of embodiment 72, comprising a step of reducing one or more interchain disulfide bonds prior to conjugating the shield moiety to AB.

[0931] 74. A method of screening masking moieties (MM) comprising:

[0932] preparing one or more variant MM;

[0933] preparing a library of one or more shield moieties to be screened, wherein a shield moiety comprises MM-CM-CS1;

[0934] preparing an antibody or fragment comprising AB-LM-CS2;

[0935] conjugating AB-LM-CS2 to one or more of MM-CM-CS1; and

[0936] determining the reduction or inhibition on AB binding elicited by said shield moiety; wherein MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;

[0937] CM represent a cleavable moiety;

[0938] EX represents an extension moiety;

[0939] LM represents a linking moiety;

[0940] CS1 represents the first part of a conjugation system;

[0941] CS2 represents the second complimentary part of said conjugation system; and AB represents an antibody or antigen binding fragment thereof.

[0942] EXAMPLES

[0943] Example 1: Selective targeting of LGR5+ cells with a-LR5-ADC

[0944] Figure 1A. demonstrates the sensitivity of cells and organoids to a-LGR5-ADC treatment. Left panel - NALM6 cells express approximately 4-fold higher levels of LGR5 than REH cells and are more sensitive to a-LGR5-ADC treatment. Right panel - LoVo CRC cells are sensitive to treatment with a-LGR5-ADC but not the non-cleavable version (a-LGR5-ADC treatmentNC). Figure 1B. Top panels - CRC organoid models (CRC1-4) have increasingly higher levels of LGR5 protein. Bottom left panel - CRC organoid models (CRC1-4) have increasingly higher levels of LGR5 protein. Bottom right panel - CRC organoid models (CRC1-4) are increasingly sensitive to a-LGR5-ADC treatment.

[0945] Figure 1C. Validation of a-LGR5-ADC efficacy in vivo using a murine model of human pre-B-ALL. Top panel - Quantification of tumour load during the treatment regime until day 18 postimplantation (PI). Lower panel - sample IVIS imaging quantifying residual tumour load at day 18 PI.

[0946] Example 2: Shield technology

[0947] Figure 2A. Top diagram -format of the Shield bearing antibody, with the antibody (pink) engaged with its epitope (pink) blue. Bottom diagram - linear format of the Shield arm. The Shield arm consists of the epitope fused to a linker arm (orange) containing protease cleavage sites and a conjugation system for attachment to the antibody. In this case the conjugation system is the SpyCatcherthat forms a covalent bond to the antibody that displays the SpyTag peptide fused to the antibody via a disulphide bridging linker. The following structure is an example of a disulphide bridging linker attached to the SpyTag, the alkyne groups present can be used to conjugate the linker to the antibody or fragment thereof:

[0948]

[0949] Figure 2B. Shielded antibody (diagram on the top left of the figure) displays less entry into LGR5 expression cells relative to the same antibody in which the Shield arm has been cleaved at its Tev cleavage site (diagram at the bottom right of the figure).

[0950] Methods: Shield Experiment

[0951] Prepare Shielded antibody:

[0952] * Incubate 2.5 pl of 50 pM spy-tagged antibody with 10x molar ratio of spy-catcher-shield (2.78 pM and 27.8 pM, respectively).

[0953] * Incubate at room temperature for 2h * For TEV cleavage: add 1 pl of 0.5 mg / ml TEV protease to 22.5 pl of antibody-shield mix and incubate for 1h

[0954] Prepare Cells:

[0955] * Plate cells at 1.5x106cells / well in a 6-well-plate (2ml / well -> 3x106cells)

[0956] o Plate NALM6, REH and 697 cells (decreasing levels of LGR5)

[0957] * Add 10 pl of 2.78 pM Shielded antibody (anti-LGR5 fused to spy-catcher shield) to each well and incubate for 15min.

[0958] FACS Preparation:

[0959] * Spin down the 2 ml of cells at 2000rpm for 5min

[0960] * Aspirate media, wash cells with 2 ml cold PBS

[0961] * Spin down cells again and aspirate the PBS

[0962] * Fix cells in 1 ml of 4% PFA in PBS and incubate for 20min

[0963] * Spin down, aspirate, wash with PBS, spin down, aspirate

[0964] * Permeabilize with 1 ml of 0.5% Triton + 2% FCS in PBS for 10 min

[0965] * Wash with 2 ml PBS, spin down, aspirate

[0966] * Block with 1 ml of 0.2% T riton + 1 % BSA + 3% goat serum and incubate for 1 h

[0967] * Wash with 2ml of PBS, spin down, aspirate

[0968] * Incubate with 500 ul 2nd antibody (antihuman IgG fused to Alexa 488) in blocking buffer (1:1000) and incubate 1h

[0969] * Wash with 2 ml PBS, spin down, aspirate

[0970] Measurement at flow cytometer

[0971] Figures 1 to 5 show results for exemplary activatable molecules tested, with binding specificity for LGR-5 and HER2 respectively. Sequence of shield arms referred to in the examples are shown below.

[0972] Sequence of shield arms containing anti-LGR5 epitope:

[0973] LGR5_1: SEQ ID NO: 161 MHHHHHHGSGGSGGASDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDF YLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGGSGSENLYFQGGSGASSGSPQGI WARGSPLGMWSRGSSGSSSSPRSGALLRGCPTHCHC LGR5_2: SEQ ID NO: 162 MHHHHHHGSGGSGGASDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDF YLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGGSGSENLYFQGGSGASSGSPQGI WARGSPLGMWSRGSSGGGGGSGGGGSGGGGSSSSSPRSGALLRGCPTHCHC

[0974] LGR5_3: SEQ ID NO: 163

[0975] MHHHHHHGSGGSGGASDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDF YLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGGSGSENLYFQGGSGASSGSPQGI WARGSPLGMWSRGSGGGGGSSGGGGGSGGGGSGGGGSSSSSPRSGALLRGCPTHCHC

[0976] CDR regions of exemplary LGR5 antibody (IMGT), note corresponding Kabat CDRs are as shown for clone 2.4

[0977] CDR H1: GYTFTNYWSEQ ID NO: 178

[0978] CDR H2: IDPSDSYT SEQ ID NO: 179

[0979] CDR H3: ARSLSGYVDY SEQ ID NO: 180

[0980] CDR L1: QDISNR SEQ ID NO: 181

[0981] CDR L2: YRS SEQ ID NO: 182

[0982] CDR L3: QQGNSLPPT SEQ ID NO: 183

[0983] Trastuzumab sequences - exemplary antibody to Her2

[0984] VH SEQ ID NO: 164

[0985] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSS

[0986] VL SEQ ID NO: 165

[0987] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIK

[0988] Heavy chain SEQ ID NO: 166

[0989] EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYAD SVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSAST KGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSL SSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPP KPKDTLMISRTPEVTCWVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLT VLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEA LHNHYTQKSLSLSPG

[0990] Light chain SEQ ID NO: 167

[0991] DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFS GSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKS GTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH KVYACEVTHQGLSSPVTKSFNRGEC

[0992] CDR regions

[0993] CDR H1: GFNIKDTYIH SEQ ID NO: 168

[0994] CDR H2: RIYPTNGYTRYADSVKG SEQ ID NO: 169

[0995] CDR H3: WGGDGFYAMDY SEQ ID NO: 170

[0996] CDR L1: RASQDVNTAVA SEQ ID NO: 171

[0997] CDR L2: SASFLYS SEQ ID NO: 172

[0998] CDR L3: QQHYTTPPT SEQ ID NO: 173

[0999] Sequence of Trastuzumab mimotope:

[1000] LLGPYELWELSHGG SEQ ID NO: 174

[1001] Sequence of shield arms containing mimotope:

[1002] H98_1: SEQ ID NO: 175

[1003] MHHHHHHGSGGSGGASDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDF YLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGGSGSENLYFQGGSGASSGSPQGI WARGSPLGMWSRGSSGSSLLGPYELWELSHGG

[1004] H98_2: SEQ ID NO: 176

[1005] MHHHHHHGSGGSGGASDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDF YLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGGSGSENLYFQGGSGASSGSPQGI WARGSPLGMWSRGSGSGGGGGSGGGGSGGGGSSSLLGPYELWELSHGG H98_3: SEQ ID NO: 177 MHHHHHHGSGGSGGASDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDF YLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGGSGSENLYFQGGSGASSGSPQGI WARGSPLGMWSRGSGGGGGSSGGGGGSGGGGSGGGGSSSLLGPYELWELSHGG

Claims

CLAIMS1. An activatable binding molecule, comprising the structure of formula (I):MM-CM-EX-LM-AB (I)wherein AB represents an antibody or antigen binding fragment thereof capable of binding a target when the binding molecule is in an activated state;MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;CM represent a cleavable moiety;EX represents an extension moiety;LM represents a linking moiety wherein the linking moiety is covalently attached via one or more amino acid side chain present in the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

2. The activatable binding molecule according to claim 1, wherein the LM is attached via one or more amino acid side chain and / or glycan, present in the CH1 domain, CL domain, hinge domain and / or Fc domain of AB, optionally wherein the amino acid side chain comprises the side chain of a naturally occurring amino acid, a genetically engineered amino acid or a genetically engineered unnatural amino acid.

3. The activatable binding molecule according to claim 1 or 2, wherein the linking moiety, LM, is attached via one or more thiol groups of cysteine residues.

4. The activatable binding molecule according to any one of claims 1 to 3, wherein the LM comprises and interchain linker.

5. The activatable binding molecule according to any one of claims 1 to 4, wherein the LM is attached to AB at one or more interchain disulfide position.

6. The activatable binding molecule according to any one of claims 1 to 5, wherein the linking moiety LM comprises and interchain disulphide re-bridging linker.

7. The activatable binding molecule according to any one of claims 1 to 6, wherein the LM comprises between 1 and 4 re-bridging moieties comprising the structure of formula (II)4;(II)wherein:ZAis a functional linking group;Pep indicates the position where the re-bridging moiety is attached to the AB, such that the re-bridging moiety re-bridges a reduced interchain disulfide bond in the ABindicates the position where the re-bridging moiety is attached to a further moiety.

8. The activatable binding molecule according to any one of claims 1 to 7, wherein the LM comprises between 1 and 4 re-bridging moieties comprising the structure of formula (Ila):wherein:one or two of A1, A2, and A3are N and the other one or two of A1, A2, and A3are CH, or all three of A1, A2, and A3are N or C;a and b are integers selected from 0 or 1;X is selected from N, NRN, O and S, wherein RNis H or C1-2 alkyl;Pep indicates where the re-bridging moiety is attached to AB.

9. The activatable binding molecule according to any one of claims 1 to 8, wherein the LM comprises between 1 and 4 re-bridging moieties comprising the structure of formula (Illa):wherein two of A1, A2and A3are N and the other of A1, A2and A3is CH;X is selected from N, O and S;Pep indicates where the re-bridging moiety is attached to AB.

10. The activatable binding molecule according to any one of claims 8 to 9, wherein A1and A2are N.

11. The activatable binding molecule according to any one of claims 8 to 10, wherein A1and A3are N.

12. The activatable binding molecule according to any one of claims 8 to 11, wherein X is selected from NRN, O and S, where RNis H or C1-2 alkyl, and there is an extension moiety, EX, attached to X via a linker.

13. The activatable binding molecule according to any one of claims 8 to 12, wherein the LM comprises the moiety of formula (lllb):wherein Q1is:where a1 = 0 to 5, b1 = 0 to 16, d = 0 to 5, d1 is 0 to 16, and b1 + d1 = 0 to 16;and YLis a functional linking moiety.

14. The activatable binding molecule according to claim 13, wherein YLis selected from the group consisting of:(b)15. The activatable binding molecule according to any one of claims 8 to 14, wherein the LM comprises the moiety of formula (lllc):where a2 = 0 to 5, b2 = 0 to 16, c2 = 0 to 5, d2 is 0 to 16, and b2 + d2 = 0 to 16.

16. The activatable binding molecule according to any one of claims 8 to 15, wherein the LM comprises the moiety of formula (IIId):Where Q3is:wherein Q4is a single bond, or 0, where Qxis such that Q4is an amino-acid residue, a dipeptide residue or a tripeptide residue, and L is a group for attachment to the extension moiety, EX.

17. The activatable binding molecule according to claim 16, wherein L is selected from:(a) a single bond;(b) -C(=0)-;(c) -NH-; and(d)18. The activatable binding molecule according to any one of claims 8 to 17, wherein the LM is of formula (IIIe):where D is the extension moiety EX.

19. The activatable binding molecule according to any one of claims 1 to 18, wherein the LM or AB is conjugated to further moiety.

20. The activatable binding molecule according to claim 19, wherein the further moiety is a labelling moiety.

21. The activatable binding molecule according to claim 20, wherein the labelling moiety is a fluorophore, a fluorescer, a radiolabel, a chemiluminescer, a nuclear magnetic resonance active label, a photosensitizer or a biotin tag.

22. The activatable binding molecule according to claim 19, wherein the further moiety is a cell killing agent, an immune-modulating payload, a macrophage class switching agent, antisense oligonucleotide or a light activatable payload.

23. The activatable binding molecule according to claim 22, wherein the immune-modulating payload is a STING agonist or a toll-like receptor agonist.

24. The activatable binding molecule according to claim 22, wherein the cell killing agent comprises a cytotoxin.

25. The activatable binding molecule according to claim 22, wherein said cytotoxin is selected from:i) a peptide toxin; orii) a chemical toxin.

26. The activatable binding molecule according to claim 24 or 25, wherein the cytoxin is selected from the group comprising auristatins, maytansinoids, tubulysins, RNA polymerase II inhibitors, transcription inhibitors, calicheamicins, duocarmycins, pyrrolobenzodiazepines, camptothecin analogues, topoisomerase inhibitors and doxorubicin.

27. The activatable binding molecule according to any one of claims 8 to 26, wherein X is N and there is one active agent and one extension moiety EX, each attached to X via a linker.

28. The activatable binding molecule according to claim 27, wherein the LM comprises the moiety of formula (lllf):wherein Q1and YLare as defined in either claims 13 or 14;Q5is:where a5 = 0 to 5, b5 = 0 to 16, c5 = 0 to 5, d5 is 0 to 16, and b5 + d5 = 0 to 16;and YL2is a functional linking moiety.

29. The activatable binding molecule according to claim 28, wherein YL2is selected from the group consisting of:(b) -C(=0)NH-30. The activatable binding molecule according to any one of claim 27 to 29, wherein the LM comprises the moiety of formula (Illg):wherein Q2is as defined in claim 15;Q6is:where a6 = 0 to 5, b6 = 0 to 16, c6 = 0 to 5, d6 is 0 to 16, and b6 + d6 = 0 to 16.

31. The activatable binding molecule according to any one of claim 27 to 30, wherein the LM comprises the moiety of formula (lllh):wherein Q3is as defined in either claim 16 or 17;Q7is:A Ms. Awherein Q8is a single bond, or O, where Qxis such that Q4is an amino-acid residue, a dipeptide residue or a tripeptide residue, and L2is a group for attachment to an active agent or EX.

32. The activatable binding molecule according to claim 31, wherein L2is selected from:(a) a single bond;(b) -C(=0)-;(c) -NH-; and(d)33. The activatable binding molecule according to any one of claim 27 to 32, wherein the LM is of formula (Illi):where D is the active agent and D2 is the extension moiety EX.

34. The activatable binding molecule according to any one of claims 7 to 33, wherein the LM comprises Formula (IVA), shown below:Z1Q1Z1- Q1- Q2- Q3- (W1)r- (W2)s- Q3- Q1- Z1Q1Z1(Formula IVA)wherein:Z1is a re-bridging moiety of Formula (II) as defined in any one of claims 7 to 9 above that is attached to an antibody at the positions shown in Formula (II); each Q1is independently a bond or a group of Formula Va:Formula IVawherein:Q1Ais absent or selected from a C(=O), OC(=O), NR7C(=O), C(=NR8) and C(=S);Q1Bis selected from O, N, S and CR9CR10;R7, R8, R9and R10are independently selected from hydrogen and C1-C4alkyl;R20and R21are independently selected from a bond, a C1-C10 alkylene and a C1-C10 alkylene containing O in the backbone; andv is an integer selected from 0, 1 or 2;each Q2is independently selected from N and CR11, wherein R11is selected from hydrogen and C1-C4alkyl;each Q3is independently a group of Formula Vb:Formula Vbwherein:Q3Ais selected from NR12, O and S;Q3Band Q3Care independently selected from a bond, O and NR13; R12and R13are independently selected from hydrogen and C1-C4alkyl; R22and R23are independently selected from a bond, a C1-C10alkylene and a C1-C10alkylene containing O in the backbone;Q3Dis absent or selected from C(=O), OC(=O), NR12C(=O) and C(=O)NR12;W1is a group of Formula Vc or Formula Ve:W1CW10“I R24R24WBW113L_ -I m — —I mR23Formula Vc Formula Ve wherein:W1Ais selected from N and CR14, wherein R14is a selected from hydrogen and C1-C4alkyl;Lwis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing amino, O or N in the backbone;Ring A is a 6-membered aryl, a 6-membered heteroaryl, a 6-membered heterocyclyl or a 6-membered cycloalkyl, wherein Xi is selected from CH or N;W1Bis a group selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR15C(=O)-, -C(=O)NR15-, -NR15C(=O)NR16-, -C(=NR15)- and -C(=S)- R15and R16are independently selected from hydrogen and C1-C4alkyl; R23and R24are independently selected from a bond, Ci-Ci2alkylene and Ci-Ci2alkylene containing O in the backbone;m is an integer selected from 1 or 2;W1Cis selected from Formula W01or Formula W02:W02FG' W01Formula W01Formula W02wherein:W01is a group of Formula W03:wherein:QW1is absent or selected from a C1-C12alkylene and a C1-C12alkylene containing O or N in the backbone; and QW2is selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - NHC(=O)-, and -C(=O)NH-;y is an integer selected from 0 or 1;XW1is selected from O or NH;XW2is selected from hydrogen, C1-C4alkyl, ORx1and NRx1Rx2, wherein Rx1and Rx2are independently selected from hydrogen and C1-C4alkyl;WQ2indicates the position where XW1is attached to a group of formula W02;R24indicates the position where W01is attached to a group of formula R24W02is a group of Formula WC4:FG' |_Q1.QW1AW31Formula WC4wherein:QW1Ais absent or selected from a C1-C12alkylene and a C1-C12alkylene containing O or N in the backbone; andQW2Ais selected from -C(=O)-, -OC(=O)-, -C(=O)O-, - NHC(=O)-, and -C(=O)NH-; LQ1is a linker comprising one or more groups selected from a C1-C20alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereof;XW3is selected from O or NH;windicates the position where XW4is attached to another group of formula W02via the QW2Asubstituent group, orWindicates the position where XW4is attached to one of the following groups: a hydrogen or - C(=O)RX3, wherein Rx3is selected from hydrogen and C1-C4alkyl;W31indicates the position whereis attached to the group of formula W01via the XW1group; XW4is selected from O or NH;LQ1is a linker comprising one or more groups selected from a C₁-C₂₀alkylene, an alkylenediamine moiety, a (poly)ethylene glycol moiety, an amino acid residue and combinations thereoft is an integer selected from 1 to 8;FG’ is a functional linking moiety;L1is a linker; andD is a further moiety selected from a shield arm or active agent;W2is a group of Formula Vd:R25Formula IVdwherein:W2Ais selected from N and CR17;W2Bis a group selected from N, CR18, -C(=O)N- and -C(=O)CR18-; W2Cis a group selected from -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -NR17C(=O)-, -C(=O)NR17-, -NR17C(=O)NR18-, -C(=NR17)- and -C(=S)- L2Wis absent or selected from a C1-C6alkylene and a C1-C6alkylene containing amino, O or N in the backbone;R17and R18are independently selected from hydrogen and C1-C4alkyl; R25and R26are independently selected from a bond, C₁-C₆alkylene and C₁-C₆alkylene containing O in the backbone; and FG’, L1and D are as defined above;r is an integer selected from 0 to 12;s is an integer selected from 0 to 6; andwherein r and s are selected such that the total number of active agents per conjugate is between 1 and 12.

35. The activatable binding molecule according to any one of claims 1 to 34, wherein the antigen binding fragment is selected from a scFv, Fab, Fab’or F(ab’)2 fragment.

36. The activatable binding molecule according to any one of claims 1 to 35, wherein the masking moiety, MM, comprises an amino acid sequence that binds to AB and inhibits the interaction of AB with the target.

37. The activatable binding molecule according to claim 36, wherein the amino acid sequence that binds comprises an epitope which binds to AB, optionally wherein the epitope comprises one or more amino acid substitution which modulates the affinity of the epitope to AB.

38. The activatable binding molecule according to any one of claims 1 to 35, wherein the MM comprises an amino acid sequence that sterically hinders the interaction of AB with the target.

39. The activatable binding molecule according to any one of claims 1 to 38, wherein the cleavable moiety, CM, is enzymatically cleavable or acid labile.

40. The activatable binding molecule according to claim 39, wherein the CM is protease cleavable.

41. The activatable binding molecule according to claim 40, wherein the protease is selected from a metalloprotease, a threonine protease, a serine protease, an aspartic acid protease, or a cysteine protease42. The activatable binding molecule according to claim 39 or 40, wherein the protease is selected from a matrix metalloproteinase (MMP), cathepsin, a kallikrein, a disintegrin and metalloproteinase (ADAM), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMts), Granzyme B.

43. The activatable binding molecule according to claim 42, wherein the MMP is selected from MMP-1, -3, -7, -8, -10, -12, -13, -20, -27, -2, -9, -11, -17, -25, -14, -15, -16, -24, -19, -21, -23, -26, and / or -28.

44. The activatable binding molecule according to any one of claims 1 to 43, wherein the extension moiety, EM, comprises a PEG linker, polysarcosine linker and / or a Gly / Ser linker second linking moiety.

45. The activatable binding molecule according to any one of claims 1 to 44, wherein the EM comprises a conjugation system, which allows for conjugation of MM-CM to LM-AB.

46. The activatable binding molecule according to claim 45, wherein the conjugation system comprises a catcher-tag system.

47. The activatable binding molecule according to claim 46, wherein the catcher-tag system is selected from SpyCatcher-SpyTag system, SnoopTag-SnoopCatcher system, Spy-Stapler-SpyTag system, Spyligase-SpyTag system, SdyCatcher-SpyTag system, SilkCatcher-SilkTag, N1 / 2-C1 / 2 system, N4 / 5-C4 / 5 system, N5 / 6-C5 / 6 system, N6 / 7b-C6 / 7b system, CnaB2Ctacher-CnaB2Tag system, PilinC-lsopeptagC system, PilinN-lsopeptagN system, DogCatcher-DogTag system, 4oq1c- 4oq1Tsystem, 3kptCc- 3kptCT, Snoopligase- SnoopTagJr, Jo-In system.

48. The activatable binding molecule according to claim 45, wherein click chemistry is used to conjugate MM-CM to LM-AB.

49. The activatable binding molecule according to claim 48, wherein the click chemistry is used to conjugate together MM-CM to LM-AB is selected from an inverse electron demand Diels-Alder (iEDDA), a copper catalyzed azide-alkyne cycloaddition (CuAAC) reaction and / or a strained-promoted azide-alkyne click chemistry (SPAAC) reaction.

50. The activatable binding molecule according to any one of claims 1 to 49, wherein AB binds to LRG5.

51. The activatable binding molecule according to any one of claim 1 to 50, wherein AB comprises and antibody or antigen binding fragment thereof that binds to LRG5.

52. The activatable binding molecule according to any one of claim 50 or 51, wherein AB binds an epitope located within amino acids 22-37 of SEQ ID NO.1.

53. The activatable binding molecule according to any one of claim 50 to 52, wherein AB binds an epitope which consists of amino acids 22-37 of SEQ ID NO.1.

54. The activatable binding molecule according to any one of claim 50 to 53, wherein the VH of the antibody comprises the following CDR1, CDR2 and CDR3:a) a CDR1 of SEQ ID No. 50 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 51 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 52 or a sequence with at least 90% homology thereto; orb) a CDR1 of SEQ ID No. 2 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 3 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 4 or a sequence with at least 90% homology thereto; orc) a CDR1 of SEQ ID No. 8 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 9 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 10 or a sequence with at least 90% homology thereto; ord) a CDR1 of SEQ ID No. 14 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 15 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 16 or a sequence with at least 90% homology thereto; ore) a CDR1 of SEQ ID No. 20 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 21 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 22 or a sequence with at least 90% homology thereto; orf) a CDR1 of SEQ ID No. 26 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 27 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 28 or a sequence with at least 90% homology thereto; org) a CDR1 of SEQ ID No. 32 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 33 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 34 or a sequence with at least 90% homology thereto; orh) a CDR1 of SEQ ID No. 38 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 39 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 40 or a sequence with at least 90% homology thereto; ori) a CDR1 of SEQ ID No. 44 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 45 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 46 or a sequence with at least 90% homology thereto; orj) a CDR1 of SEQ ID No. 56 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 57 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 58 or a sequence with at least 90% homology thereto; ork) a CDR1 of SEQ ID No. 62 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 63 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 64 or a sequence with at least 90% homology thereto; orl) a CDR1 of SEQ ID No. 68 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 69 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 70 or a sequence with at least 90% homology thereto; orm) a CDR1 of SEQ ID No. 74 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 75 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 76 or a sequence with at least 90% homology thereto.

55. An activatable binding molecule according to any one of claim 50 to 54, wherein the VL of the antibody comprises the following CDR1, CDR2 and CDR3:a) a CDR1 of SEQ ID No. 53 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 54 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 55 or a sequence with at least 90% homology thereto; orb) a CDR1 of SEQ ID No. 5 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 6 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 7 or a sequence with at least 90% homology thereto; orc) a CDR1 of SEQ ID No. 11 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 12 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 13 or a sequence with at least 90% homology thereto; ord) a CDR1 of SEQ ID No. 17 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 18 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 19 or a sequence with at least 90% homology thereto; ore) a CDR1 of SEQ ID No. 23 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 24 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 25 or a sequence with at least 90% homology thereto; orf) a CDR1 of SEQ ID No. 29 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 30 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 31 or a sequence with at least 90% homology thereto; org) a CDR1 of SEQ ID No. 35 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 36 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 37 or a sequence with at least 90% homology thereto; orh) a CDR1 of SEQ ID No. 41 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 42 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 43 or a sequence with at least 90% homology thereto; ori) a CDR1 of SEQ ID No. 47 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 48 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 49 or a sequence with at least 90% homology thereto; orj) a CDR1 of SEQ ID No. 59 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 60 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 61 or a sequence with at least 90% homology thereto; ork) a CDR1 of SEQ ID No. 65 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 66 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 67 or a sequence with at least 90% homology thereto; orl) a CDR1 of SEQ ID No. 71 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 72 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 73 or a sequence with at least 90% homology thereto; orm) a CDR1 of SEQ ID No. 77 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 78 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 79 or a sequence with at least 90% homology thereto.

56. The activatable binding molecule according to any one of claim 50 to 55, comprising a VH sequence selected from SEQ ID NOs: 96, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104.

57. The activatable binding molecule according to any one of claim 50 to 56, comprising a VL sequence selected from SEQ ID NOs: 97, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105.

58. The antibody or fragment thereof according to any one of claim 50 to 57 comprising: i) a VH sequence of SEQ ID NO: 96 and a VL sequence of SEQ ID NO: 97;b) a VH sequence of SEQ ID NO: 80 and a VL sequence of SEQ ID NO: 81;c) a VH sequence of SEQ ID NO: 82 and a VL sequence of SEQ ID NO: 83;d) a VH sequence of SEQ ID NO: 84 and a VL sequence of SEQ ID NO: 85;e) a VH sequence of SEQ ID NO: 86 and a VL sequence of SEQ ID NO: 87;f) a VH sequence of SEQ ID NO: 88 and a VL sequence of SEQ ID NO: 89;g) a VH sequence of SEQ ID NO: 90 and a VL sequence of SEQ ID NO: 91;h) a VH sequence of SEQ ID NO: 92 and a VL sequence of SEQ ID NO: 93;i) a VH sequence of SEQ ID NO: 94 and a VL sequence of SEQ ID NO: 95;j) a VH sequence of SEQ ID NO: 98 and a VL sequence of SEQ ID NO: 99;k) a VH sequence of SEQ ID NO: 100 and a VL sequence of SEQ ID NO: 101l) a VH sequence of SEQ ID NO: 102 and a VL sequence of SEQ ID NO: 103; orm) a VH sequence of SEQ ID NO: 104 and a VL sequence of SEQ ID NO: 105.

59. The activatable binding molecule according to any one of claim 50 to 58, comprising a sequence of an antibody clone as shown in Table 3 or a sequence with at least 70%, 80% or 90% homology thereto.

60. The activatable binding molecule according to claim 50 or 59 wherein the fragment is an scFV comprising SEQ ID NO. 211 or a sequence with at least 70%, 80% or 90% homology thereto.

61. The activatable binding molecule according to any one of claim 50 to 53, wherein AB comprises and antibody or antigen binding fragment thereof that binds to LRG5, wherein the VH of the antibody or antigen binding fragment comprises the following CDR1, CDR2 and CDR3: a) a CDR1 of SEQ ID No. 50 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 51 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 52 or a sequence with at least 90% homology thereto; orb) a CDR1 of SEQ ID No. 2 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 3 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 4 or a sequence with at least 90% homology thereto; orc) a CDR1 of SEQ ID No. 8 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 9 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 10 or a sequence with at least 90% homology thereto; ord) a CDR1 of SEQ ID No. 14 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 15 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 16 or a sequence with at least 90% homology thereto; ore) a CDR1 of SEQ ID No. 20 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 21 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 22 or a sequence with at least 90% homology thereto; orf) a CDR1 of SEQ ID No. 26 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 27 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 28 or a sequence with at least 90% homology thereto; org) a CDR1 of SEQ ID No. 32 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 33 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 34 or a sequence with at least 90% homology thereto; orh) a CDR1 of SEQ ID No. 38 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 39 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 40 or a sequence with at least 90% homology thereto; ori) a CDR1 of SEQ ID No. 44 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 45 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 46 or a sequence with at least 90% homology thereto; orj) a CDR1 of SEQ ID No. 56 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 57 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 58 or a sequence with at least 90% homology thereto; ork) a CDR1 of SEQ ID No. 62 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 63 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 64 or a sequence with at least 90% homology thereto; orl) a CDR1 of SEQ ID No. 68 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 69 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 70 or a sequence with at least 90% homology thereto; orm) a CDR1 of SEQ ID No. 74 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 75 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 76 or a sequence with at least 90% homology thereto.

62. The activatable binding molecule according to claim 61 which binds to LGR5 wherein the VL of the antibody comprises the following CDR1, CDR2 and CDR3:a) a CDR1 of SEQ ID No. 53 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 54 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 55 or a sequence with at least 90% homology thereto; orb) a CDR1 of SEQ ID No. 5 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 6 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 7 or a sequence with at least 90% homology thereto; orc) a CDR1 of SEQ ID No. 11 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 12 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 13 or a sequence with at least 90% homology thereto; ord) a CDR1 of SEQ ID No. 17 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 18 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 19 or a sequence with at least 90% homology thereto; ore) a CDR1 of SEQ ID No. 23 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 24 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 25 or a sequence with at least 90% homology thereto; orf) a CDR1 of SEQ ID No. 29 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 30 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 31 or a sequence with at least 90% homology thereto; org) a CDR1 of SEQ ID No. 35 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 36 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 37 or a sequence with at least 90% homology thereto; orh) a CDR1 of SEQ ID No. 41 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 42 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 43 or a sequence with at least 90% homology thereto; ori) a CDR1 of SEQ ID No. 47 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 48 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 49 or a sequence with at least 90% homology thereto; orj) a CDR1 of SEQ ID No. 59 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 60 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 61 or a sequence with at least 90% homology thereto; ork) a CDR1 of SEQ ID No. 65 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 66 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 67 or a sequence with at least 90% homology thereto; orl) a CDR1 of SEQ ID No. 71 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 72 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 73 or a sequence with at least 90% homology thereto; orm) a CDR1 of SEQ ID No. 77 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 78 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 79 or a sequence with at least 90% homology thereto.

63. The activatable binding molecule according to claim 61 or 62, comprising a VH sequence selected from SEQ ID NOs: 96, 80, 82, 84, 86, 88, 90, 92, 94, 98, 100, 102, 104.

64. The activatable binding molecule according to claim 61 to 63, comprising a VL sequence selected from SEQ ID NOs: 97, 81, 83, 85, 87, 89, 91, 93, 95, 99, 101, 103, 105.

65. The activatable binding molecule according to any of claims 50 to 64 comprising SEQ ID NO. 161, SEQ ID NO.162 or 1 SEQ ID NO. 163.

66. The activatable binding molecule according to any one of claims 1 to 49 wherein AB binds to HER2.

67. The activatable binding molecule according to claim 66, wherein AB comprises and antibody or antigen binding fragment thereof that binds to HER2.

68. The activatable binding molecule according to any one of claim 66 or 67, wherein AB binds an epitope comprising or consisting of SEQ ID NO.174.

69. The activatable binding molecule according to any one of claims 66 to 68, wherein the VH of the antibody comprises a CDR1 of SEQ ID No. 168 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 169 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 170 or a sequence with at least 90% homology thereto.

70. The activatable binding molecule according to any one of claims 66 to 69, wherein the VL of the antibody comprises a CDR1 of SEQ ID No. 171 or a sequence with at least 90% homology thereto, a CDR2 of SEQ ID No. 172 or a sequence with at least 90% homology thereto, a CDR3 of SEQ ID No. 173 or a sequence with at least 90% homology thereto.

71. The activatable binding molecule according to any of claims 66 to 70 comprising SEQ ID NO. 175, SEQ ID NO. 176 or SEQ ID NO. 177.

72. A pharmaceutical composition comprising an activatable binding molecule according to any one of claims 1 to 71 and a pharmaceutical carrier.

73. An activatable binding molecule according to any one of claims 1 to 71 or a pharmaceutical composition according to claim 72 for use as a medicament.

74. An activatable binding molecule according to any one of claim 1 to 71 or a pharmaceutical composition according to claim 72 for use in the treatment or diagnosis of a cancer or an inflammatory disease.

75. A method for treating a cancer comprising administering a therapeutically effective amount of an activatable binding molecule according to any one of claim 1 to 71, or a pharmaceutical composition according to claim 72.

76. The activatable binding molecule for use of claim 74 or the method of claim 75, wherein the cancer is an LGR5-positive cancer.

77. The activatable binding molecule for use of claim 74 or the method of claim 75 wherein the cancer overexpresses LGR5.

78. The activatable binding molecule for use of any of claim 73, 74 or 77, or the method of any of claim 75 to 77 wherein the cancer is selected from cancer of the head or neck, uterine cancer, colorectal cancer, stomach cancer, carcinoma of the endometrium, cancer of the esophagus, leukemia, such as acute lymphoblastic leukemia (ALL), liver cancer, such as hepatocellular carcinoma, or pancreatic cancer.

79. A method of preparing an activatable binding molecule comprising:conjugating a shield moiety comprising MM-CM-EX-LM- to an antibody or fragment thereof (AB), to form an activatable binding molecule,wherein the shield moiety is conjugated to AB via the CH1 domain, CL domain, hinge domain and / or Fc domain of AB.

80. The method of claim 79, comprising a step of reducing one or more interchain disulfide bonds prior to conjugating the shield moiety to AB.

81. A method of screening masking moieties (MM) comprising:preparing one or more variant MM;preparing a library of one or more shield moieties to be screened, wherein a shield moiety comprises MM-CM-CS1;preparing an antibody or fragment comprising AB-LM-CS2;conjugating AB-LM-CS2 to one or more of MM-CM-CS1; anddetermining the reduction or inhibition on AB binding elicited by said shield moiety; wherein MM represents a masking moiety capable of inhibiting or reducing interaction of AB with a target;CM represent a cleavable moiety;EX represents an extension moiety;LM represents a linking moiety;CS1 represents the first part of a conjugation system;CS2 represents the second complimentary part of said conjugation system; and AB represents an antibody or antigen binding fragment thereof.

Images