Anti-beta-catenin antibodies

Abstract

The present disclosure relates to antibodies directed against beta-catenin and formulations comprising the same. The disclosure further relates to the use of beta-catenin antibodies and formulations in therapy, particularly in the treatment of solid tumors.

Description

[Technical Field]

[0001] The present invention relates to antibodies directed against beta-catenin and formulations comprising the same. The present invention further relates to the use of beta-catenin antibodies and formulations in therapy, particularly in the treatment of solid tumors. [Background technology]

[0002] Intracellular antibodies (also called intrabodies) are antibodies or antibody fragments that are localized within specific intracellular compartments, sometimes via intracellular trafficking signals, where they interact with target antigens. To function as intrabodies, antibodies must retain stability and affinity for their targets in the reducing environment of the cell. Because antibodies rely on inter- and intrachain disulfide bonds for stability, which cannot form within the reducing environment of the cytoplasm, full-length antibodies are inherently unstable intracellularly (Biocca et al., 1995). Nevertheless, other stable options exist, including the use of engineered antibody fragments or alternative non-immunoglobulin-binding proteins. Intrabodies have proven useful as research tools for modulating intracellular target protein function (Stocks, 2005).

[0003] Camelids (Camelidae) are known to produce heavy-chain antibodies (HCAbs) as part of their adaptive immune response, in addition to antibodies containing conventional heavy and light chains. Because these HCAbs lack both the entire light chain and the CH1 domain of conventional IgG, antigen binding is mediated solely by unpaired VH domains, termed VHHs. There have been several reports of intracellularly functional VHH intrabodies in llamas (Serruys et al., 2009; Vercruysse et al., 2010). VHH domains have already been used as research intrabodies due to their inherent domain stability and low aggregation tendency.

[0004] Wnt proteins are a large family of lipid-modified secreted signaling proteins defined by their amino acid sequence rather than their functionality. They are highly conserved across the animal kingdom. Wnt signaling is initiated by binding of Wnt lipoproteins to extracellular receptors, such as the seven-transmembrane frizzled receptor family, LRP5 and six co-receptors, and the receptor tyrosine kinases Ryk and ROR. Studies of beta-catenin have demonstrated that it has at least two cellular roles: as a key transcriptional coactivator in the canonical Wnt signaling pathway and as a critical component of adherens junctions, where beta-catenin connects E-cadherin to the cytoskeleton via alpha-catenin (Ben-Ze'ev and Geiger, 1998). Activation of the canonical Wnt / b-catenin pathway by exogenous Wnt leads to the stabilization and activation of beta-catenin, which translocates from the cytoplasm to the nucleus, where it interacts with numerous partners, including the TCF / LEF family. Formation of the bipartite beta-catenin / TCF transcription factor 2 activates transcription of Wnt-responsive genes.

[0005] Abnormalities in the Wnt signaling pathway are thought to be associated with many diverse diseases, including colorectal cancer, adenomatous polyposis (FAP), colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma, pilomatricoma, and prostate cancer, as well as bone mineral density disorders, Alzheimer's disease (Morin et al., 1999; Newnham et al., 2015), schizophrenia (Miyaoka et al., 1999), type II diabetes (Grant et al., 2006), rheumatoid arthritis (Sen et al., 2000), oligodontia (Lammi et al., 2004), osteoporosis-pseudoglioma syndrome (Gong et al., 2001), familial exudative vitreoretinopathy (Toomes et al., 2004), and even vascular calcification. As a non-limiting example, the majority of colorectal cancers harbor mutations in the Wnt signaling pathway, particularly in the APC gene (Bienz and Clevers, 2000), which leads to stabilization and excessive transcriptional activity of beta-catenin.

[0006] Several antibodies targeting beta-catenin have already been disclosed. For example, McCrea (1993) described a Fab fragment directed to the N-terminal residues 6-138 of beta-catenin, which can affect the developmental pattern of Xenopus embryos. WO2021015419 discloses an antibody that specifically binds to phosphorylated beta-catenin and whose epitope consists of amino acid residues 42-51 of full-length beta-catenin.

[0007] Therefore, there remains a need to provide antibodies that inhibit Wnt signaling by binding to beta-catenin and blocking the biological activity of beta-catenin, such as its transcriptional activation activity. Such antibodies would be extremely useful in therapies, including therapeutic interventions, for colorectal cancer or adenomatous polyps. In particular, there remains a need to provide anti-beta-catenin antibodies that function intracellularly as intrabodies to disrupt the transcriptional activation activity of beta-catenin. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention addresses the above-identified needs by providing novel antibodies against beta-catenin. These antibodies may be useful in therapy, particularly in the treatment of colorectal cancer or adenomatous polyps. These antibodies have particularly high specificity for beta-catenin and specifically disrupt the co-transcriptional activity of beta-catenin without affecting its role at the plasma membrane. [Means for solving the problem]

[0009] In a first aspect, the present invention provides an antibody that specifically binds to human beta-catenin, the antibody comprising: i) a CDR-H1 comprising SEQ ID NO: 1, 35, or 36; a CDR-H2 comprising SEQ ID NO: 2, 37, or 38, and a CDR-H3 comprising SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii) a CDR-H1 comprising SEQ ID NO: 4, a CDR-H2 comprising SEQ ID NO: 5, and a CDR-H3 comprising SEQ ID NO: 6 or 46; or iv) a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of i to iii. Preferably, the antibody is a VHH.

[0010] In a second aspect, the present invention provides an antibody that specifically binds to human beta-catenin, the antibody comprising: i) an FR1 comprising SEQ ID NO: 10, 15, or 18, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and ii) an FR1 comprising SEQ ID NO: 11, 16, or 19, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. iii) FR3 comprising SEQ ID NO: 12, 13, 17, or 20, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and iv) FR4 comprising SEQ ID NO: 14, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. Preferably, the antibody is a VHH.

[0011] In a third aspect, the present invention provides an antibody that specifically binds to human beta-catenin, wherein the antibody has a heavy chain variable region comprising any one of SEQ ID NOs: 21 to 25, 28, 31, or 33, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0012] Further aspects of the invention include isolated polynucleotides encoding the antibodies, cloning or expression vectors, host cells, processes for the production of the antibodies, pharmaceutical compositions comprising the antibodies, and their uses in therapy. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present disclosure will now be described with respect to certain non-limiting aspects and embodiments thereof, with reference to certain figures and examples.

[0014] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the subject matter of this specification belongs. As used in the specification and claims, the following definitions are provided to facilitate understanding of the invention.

[0015] - The term "and / or" as used herein in phrases such as "A and / or B" is intended to include "A and B," "A or B," "A," and "B."

[0016] - The forms "a," "an," and "the" include both singular and plural referents unless the content clearly dictates otherwise. Thus, for example, a reference to "an antibody" includes "one antibody" and "antibodies."

[0017] - the term "comprising" does not exclude other elements. For the purposes of the present disclosure, the term "consisting of" is considered to be a preferred embodiment of the term "comprising".

[0018] - The term "beta-catenin" refers to a polypeptide that is involved in the Wnt pathway and cell-cell adhesion, among other functions. The amino acid and nucleic acid sequences of beta-catenin and its isoforms are also well known in the art (see, for example, UNIPROT P35222). Complete human beta-catenin comprises the sequence given in SEQ ID NO: 51. The term also encompasses any alternative splicing variants or naturally occurring variants of human beta-catenin that are naturally expressed by cells.

[0019] - The term "antibody" refers to whole antibodies and functionally active fragments thereof (i.e., molecules comprising an antigen-binding domain that specifically binds to an antigen, also referred to as antigen-binding fragments). Unless the context dictates otherwise, the characteristics described herein with respect to antibodies also apply to antibody fragments. Whole antibodies, also known as "immunoglobulins (Ig)," generally relate to intact or full-length antibodies, i.e., antibodies comprising two heavy chain and two light chain components interconnected by disulfide bonds and assembled to define a characteristic Y-shaped three-dimensional structure. Classical, naturally occurring whole antibodies are monospecific, in the sense that they bind to one antigen type, and bivalent, in the sense that they have two independent antigen-binding domains.

[0020] The terms "unmodified antibody," "full-length antibody," and "whole antibody" are used interchangeably and refer to a monospecific, bivalent antibody that contains an Fc region as defined herein and has a structure similar to that of a native antibody. In a whole antibody, each light chain consists of a light chain variable region (abbreviated herein as XL) and a light chain constant region (CL). Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (CH) that is composed of three constant domains CH1, CH2, and CH3, or four constant domains CH1, CH2, CH3, and CH4, depending on the Ig class. The "class" of an Ig or antibody refers to the type of constant region and includes IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses, e.g., IgG1, IgG2, IgG3, and IgG4. The constant regions of antibodies can mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions of antibodies according to the present invention can be further subdivided into regions of hypervariability that determine antigen recognition, termed complementarity-determining regions (CDRs) (or "hypervariable regions" or HVRs), interspersed among more structurally conserved regions termed framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The CDRs and FRs together form the variable region. By convention, the CDRs of the heavy chain variable region of an antibody or antigen-binding fragment thereof are referred to as CDR-H1, CDR-H2, and CDR-H3, and of the light chain variable region as CDR-L1, CDR-L2, and CDR-L3, numbered sequentially from the N-terminus to the C-terminus of each chain.

[0021] CDRs are conventionally numbered according to the system devised by Kabat et al., which is described in Kabat et al., 1991. This numbering system is used herein except where otherwise indicated.

[0022] The CDRs of the heavy chain variable domain typically comprise residues 31-35 (CDR-H1), residues 50-65 (CDR-H2), and residues 95-102 (CDR-H3) according to the Kabat numbering system. However, according to Chothia (1987), the loop equivalent to CDR-H1 extends from residue 26 to residue 32. Thus, unless otherwise indicated, "CDR-H1" as employed herein is intended to mean residues 26-35 as described by a combination of the Kabat numbering system and Chothia's topological loop definition. The CDRs of the light chain variable domain are typically located at residues 24-34 (CDR-L1), residues 50-56 (CDR-L2), and residues 89-97 (CDR-L3) according to the Kabat numbering system. In addition to the CDR loops, a fourth loop formed by framework 3 (FR3) exists between CDR-2 (CDR-L2 or CDR-H2) and CDR-3 (CDR-L3 or CDR-H3). The Kabat numbering system defines framework 3 as positions 66-94 of the heavy chain and positions 57-88 of the light chain.

[0023] The terms "constant domain(s)" or "constant region" as used herein are used interchangeably to refer to the domain(s) of an antibody outside the variable region. The constant domains are identical in all antibodies of the same isotype, but vary from isotype to isotype. Typically, the constant region of a heavy chain comprises three or four constant domains, formed from the N- to C-terminus by CH1-hinge-CH2-CH3-optionally CH4.

[0024] The constant region domains of the antibody molecules of the present invention may be selected with regard to the intended function of the antibody molecule and, in particular, any effector functions that may be required, if any. For example, the constant region domains may be human IgA, IgD, IgE, IgG, or IgM domains. In particular, when the antibody molecule is intended for therapeutic use and antibody effector functions are required, human IgG constant region domains, particularly of the IgG1 and IgG3 isotypes, may be used. Alternatively, when the antibody molecule is intended for therapeutic purposes and antibody effector functions are not required, IgG2 and IgG4 isotypes may be used. It will be recognized that sequence variants of these constant region domains may also be used. For example, the IgG4 molecule described in Angal et al. (Angal et al., 1993) in which the serine at position 241 (numbered according to the Kabat numbering system) has been changed to proline.

[0025] "Fc," "Fc fragment," and "Fc region" are used interchangeably and refer to the C-terminal region of an antibody that contains the constant region of the antibody excluding the first constant region immunoglobulin domain. That is, Fc refers to the C-terminal region of an antibody that contains the last two constant domains of IgA, IgD, and IgG, i.e., the C-terminal region of an antibody. H2 and C H3 The term "constant region" refers to the last three constant domains of IgE and IgM, as well as the flexible hinge N-terminal to these domains. The human IgG1 heavy chain Fc region is defined herein to include residue C226 to its carboxy terminus, where numbering is according to the EU index as in Kabat. For human IgG1, the lower hinge refers to positions 226-236, the CH2 domain refers to positions 237-340, and the CH3 domain refers to positions 341-447, according to the EU index as in Kabat. Corresponding Fc regions of other immunoglobulins can be identified by sequence alignment. In the context of the present disclosure, the constant region or Fc region, if present, may be native, as defined above, or may be modified in various ways, provided that it contains a functional FcR-binding domain, preferably a functional FcRn-binding domain.

[0026] - the term "antibody" encompasses monovalent antibodies, i.e., antibodies that comprise only one antigen-binding domain (e.g., a single-armed antibody comprising a full-length heavy chain and a full-length light chain linked to each other, also called a "half-antibody"), and multivalent antibodies, i.e., antibodies that comprise two or more antigen-binding domains.

[0027] - the term "antibody" according to the present invention also encompasses antigen-binding fragments of antibodies, including single chain antibodies (e.g. scFv and dsscfv), Fab, Fab', F(ab')2, Fv, single domain antibodies or nanobodies (e.g. VH or VL, or VHH or VNAR).

[0028] As used herein, the term "Fab fragment" refers to an antibody fragment comprising a light chain fragment containing the VL (variable light) domain and constant domain (CL) of the light chain, and a VH (variable heavy) domain and the first constant domain (CH1) of the heavy chain. A typical "Fab' fragment" comprises a heavy and light chain pair, where the heavy chain contains the variable domain VH, the constant domain CH1, and a native or modified hinge region, and the light chain contains the variable domain VL and the constant domain CL. Dimers of Fab' according to the present disclosure create F(ab')2, where, for example, dimerization may be via the hinge.

[0029] - As used herein, the term "single domain antibody" refers to an antibody fragment consisting of a single monomeric variable antibody domain. Examples of single domain antibodies include VH, VL, VHH, or VNAR.

[0030] - the term "HCab" means an antibody comprising or consisting of a VHH domain linked directly or via a linker (hinge) to a CH2 and CH3 domain.

[0031] - the term "Fv" refers to an associated pair of two variable domains, eg co-operating variable domains, eg affinity matured variable domains, ie a VH and VL pair.

[0032] - The term "single-chain variable fragment" or "scFv" as used herein means a single-chain variable fragment stabilized by a peptide linker between the VH and VL variable domains.

[0033] As used herein, the term "multispecific" or "multispecific antibody" refers to an antibody as described herein having at least two binding domains, i.e., two or more binding domains, e.g., two or three binding domains, which at least two binding domains independently bind to two different antigens or two different epitopes on the same antigen. Multispecific antibodies are generally monovalent for each specificity (antigen). Multispecific antibodies as described herein encompass monovalent and multivalent, e.g., bivalent, trivalent, tetravalent, multispecific antibodies.

[0034] - As used herein, the term "antigen-binding domain" refers to a part of an antibody that comprises part or all of one or more variable domains, e.g., part or all of a pair of variable domains VH and VL, that specifically interact with a target antigen. A binding domain may constitute a single-domain antibody. In one embodiment, each binding domain is monovalent. Preferably, each binding domain comprises only one VH and one VL.

[0035] The term "chimeric" antibody refers to an antibody in which the heavy and / or light chain variable domains (or at least a portion thereof) are derived from a particular source or species, e.g., mouse, rat, rabbit, or similar, and the remainder of the heavy and / or light chain (i.e., the constant regions) are derived from another species, such as human. Chimeric antibodies are composed of components derived from two different species, thereby retaining the characteristics of the original species from which they were derived. A subcategory of "chimeric antibodies" is "humanized antibodies." Humanized antibodies (including CDR-grafted antibodies) are antibody molecules having one or more complementarity-determining regions (CDRs) derived from a non-human species and framework regions derived from a human immunoglobulin molecule. It will be appreciated that it is not necessary to transfer the entire CDR, but only the specificity-determining residues of the CDR (Kashmiri et al., 2005, Methods, 36, pp. 25-34). Humanized antibodies may optionally further comprise one or more framework residues derived from the original non-human species from which the CDRs were derived.

[0036] - The term "fully human" antibody means an antibody in which the variable and constant regions (if present) of both the heavy and light chains are all of human origin or are substantially identical to sequences of human origin, but not necessarily derived from the same antibody. Examples of fully human antibodies can include, for example, antibodies produced by the phage display method described above, and antibodies produced in mice, in which the variable and optionally constant region genes of the mouse immunoglobulin have been replaced by their human counterparts.

[0037] Within the context of the present invention, the term "epitope" is used interchangeably to refer to both conformational epitopes, which consist of discontinuous stretches of the primary amino acid sequence of an antigen, and linear epitopes, which are formed by sequences formed by contiguous amino acids.

[0038] - The term "isolated" antibody means an antibody that has been separated (eg, by purification means) from a component of its natural environment.

[0039] - The term "isolated" polynucleotide means that the polynucleotide is present in a physical environment that is distinct from the physical environment in which it naturally occurs.

[0040] As used herein, the term "K D " is K a K d The ratio of (i.e., K d / K a ) and is expressed as a molar concentration (M). d and K. a and K respectively refer to the dissociation rate and association rate of a specific antigen-antibody (or antigen-binding fragment thereof) interaction. D The values ​​can be determined using methods well established in the art, such as (but not limited to) the methods used in the Examples section.

[0041] - The terms "blocking," "block," and the like, in reference to an antibody, describe an antibody that is capable of inhibiting or attenuating at least one biological activity of its target (beta-catenin). Alternatively, the terms "neutralizing" or "neutralize" can be used.

[0042] The term "specifically binds" refers to an antibody that binds preferentially or with high affinity to a protein of interest (e.g., beta-catenin) but does not substantially bind to other proteins. In other words, the antibody binds to the protein of interest without significant cross-reactivity with any other molecules. The specificity of an antibody can be further examined by determining whether it binds to other related proteins discussed above, or whether the antibody discriminates between proteins.

[0043] As used herein, the terms "treatment," "treating," and the like refer to obtaining a desired pharmacological and / or physiological effect. The effect may be prophylactic, in terms of completely or partially preventing a disease or its symptoms, and / or therapeutic, in terms of partially or completely curing a disease and / or adverse effects resulting from a disease. That is, "treatment" encompasses any treatment of a disease in a mammal, particularly a human, including (a) preventing a disease from occurring in a subject who may be affected by the disease but has not yet been diagnosed as having the disease, (b) arresting a disease, i.e., blocking its development, and (c) relieving a disease, i.e., causing regression of a disease.

[0044] - the term "therapeutically effective amount" means the amount of an active ingredient (e.g. an antibody according to the invention) that, when administered to a mammal or other subject for treating a disease, is sufficient to effect treatment for the disease.

[0045] There have been several challenges associated with providing antibodies against beta-catenin, including the fact that 1) beta-catenin is an intracellular protein, so anti-beta-catenin antibodies administered in vivo must cross the cell membrane, and 2) the target (i.e., beta-catenin) has two major but completely distinct activities, and blocking only one of these biological activities would be beneficial. In particular, as described in more detail herein, the present invention is based on the discovery of antagonistic anti-beta-catenin antibodies that can neutralize / inhibit the transcriptional activation activity of beta-catenin without affecting its activity in cell-cell adhesion.

[0046] The present invention also provides evidence that the epitopes recognized by the antibodies of the present invention are found in the native conformation(s) of intracellular beta-catenin, thereby supporting for the first time the use of anti-beta-catenin antibodies that are in VHH format and can act as intrabodies.

[0047] The primary object of the present invention is an antibody that specifically binds to beta-catenin, the antibody comprising: i. CDR-H1 of SEQ ID NO: 1, 35, or 36; CDR-H2 of SEQ ID NO: 2, 37, or 38, and CDR-H3 of SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii. CDR-H1 of SEQ ID NO: 4, CDR-H2 of SEQ ID NO: 5, and CDR-H3 of SEQ ID NO: 6 or 46; iii. CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; and CDR-H3 of SEQ ID NO: 9, 47, 48, 49, or 50; or iv. CDR-H1, CDR-H2, and CDR-H3 sequences having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of i-iii. The heavy chain variable region comprises:

[0048] As non-limiting examples, antibodies according to the invention include: a) an antibody comprising a CDR-H1 according to SEQ ID NO: 1; a CDR-H2 according to SEQ ID NO: 2; and a CDR-H3 according to SEQ ID NO: 3; b) an antibody comprising a CDR-H1 according to SEQ ID NO: 4, a CDR-H2 according to SEQ ID NO: 5, and a CDR-H3 according to SEQ ID NO: 6; c) an antibody comprising a CDR-H1 according to SEQ ID NO: 7, a CDR-H2 according to SEQ ID NO: 8, and a CDR-H3 according to SEQ ID NO: 9; d) an antibody "V1" comprising a CDR-H1 according to SEQ ID NO: 35, a CDR-H2 according to SEQ ID NO: 37, and a CDR-H3 according to SEQ ID NO: 39; f) antibody "V2" comprising CDR-H1 according to SEQ ID NO: 35, CDR-H2 according to SEQ ID NO: 37 and CDR-H3 according to SEQ ID NO: 40; f) antibody "V3" comprising CDR-H1 according to SEQ ID NO: 36, CDR-H2 according to SEQ ID NO: 38 and CDR-H3 according to SEQ ID NO: 41; g) antibody "S" comprising CDR-H1 according to SEQ ID NO: 4, CDR-H2 according to SEQ ID NO: 5 and CDR-H3 according to SEQ ID NO: 6; or further h) antibody "O" comprising CDR-H1 according to SEQ ID NO: 7, CDR-H2 according to SEQ ID NO: 8 and CDR-H3 according to SEQ ID NO: 47.

[0049] Anti-beta-catenin antibodies according to the present invention (i.e., comprising any one of the above-mentioned CDR sequence combinations) are particularly patentable because they provide the antibodies with high affinity for human beta-catenin, high inhibitory activity against at least one biological function of beta-catenin (preferably a function in the Wnt pathway), and high stability, which is important for manufacturability.

[0050] In one embodiment of the invention, the antibody comprises: i. FR1 comprising SEQ ID NO: 10, 15, or 18, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; ii. FR2 comprising SEQ ID NO: 11, 16, or 19, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; iii. FR3 comprising SEQ ID NO: 12, 13, 17, or 20, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and iv. FR4 comprising SEQ ID NO: 14 or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. It further comprises a framework region (FR) comprising (or consisting of):

[0051] In another embodiment, the invention provides an antibody that specifically binds to beta-catenin, wherein the antibody has a heavy chain variable region comprising any one of SEQ ID NOs: 21, 22, 23, 24, 25, 28, 31, or 33, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0052] In another embodiment, the present invention provides an antibody that specifically binds to beta-catenin, wherein the antibody further comprises a CH2 and a CH3 domain. Non-limiting examples of CH2 / CH3 domains include any one of SEQ ID NOs: 55 or 56, or sequences having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The heavy chain variable region can be linked to the CH2 / CH3 domain by itself, or can be linked via a linker (also referred to as a hinge). Non-limiting examples of linkers include any one of SEQ ID NOs: 57 or 58. Non-limiting examples of HCabs comprising a heavy chain variable region and CH2 / CH3 domains with or without a linker can be selected from any one of SEQ ID NOs: 59, 60, 61, and 62, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

[0053] The antibodies according to the present invention as a whole are preferably VHH or Hcab, i.e., they are not associated with any light chain. In other words, the antibodies according to the present invention as a whole preferably consist of only one heavy chain variable domain and an active fragment thereof or only one complete heavy chain and an active fragment thereof. Since it has been shown that the antibodies according to the present invention can be active intracellularly, they are instead referred to as anti-beta-catenin intrabodies. The antibodies described herein may be chimeric or humanized. When the antibodies are humanized, suitable framework regions for the heavy chains of the humanized antibodies according to the present invention can be derived from human germline. They are preferably in VHH or Hcab format (more preferably VHH format), but may instead be accompanied by a light chain comprising CDRs specifically binding to beta-catenin. Alternatively, to obtain bispecific or multispecific antibodies, they may be accompanied by a light chain comprising CDRs specifically binding to one or more other targets.

[0054] Overall, the anti-beta-catenin antibodies of the present invention are antagonistic antibodies. Preferably, they neutralize / inhibit at least one of the biological activities of beta-catenin. Preferably, at least one biological activity of beta-catenin that is neutralized / inhibited is transcriptional activation activity. More preferably, only one biological activity of beta-catenin, i.e., transcriptional activation activity, is neutralized / inhibited. As a specific example, the anti-beta-catenin antibodies i) block the binding of any one or more of Pontin52, Bcl-9, and lymphoid enhancer factor 1 (LEF-1) / T-cell factor (TCF) family (TCF) to beta-catenin, and / or ii) neutralize or inhibit a pathway mediated by beta-catenin, such as the transcriptional activation activity of beta-catenin. In another example, the anti-beta-catenin antibodies described herein prevent the interaction of any one or more of Pontin52, Bcl-9, and lymphoid enhancer factor 1 (LEF-1) / T-cell factor (TCF) family members (TCFs) with beta-catenin, thereby inhibiting the transcriptional activation activity of beta-catenin.

[0055] Overall, the anti-beta-catenin antibodies of the present invention preferably have an equilibrium dissociation constant (KD) for beta-catenin of 20 nM or less, preferably 15 nM or less, more preferably 10 nM or less, for example 9 nM or less, 8 nM or less, or 7 nM or less. In certain embodiments, the KD is as low as 6 nM or less, 5 nM or less, or 4 nM or less. The KD can be measured / determined by any suitable method. For example, the dissociation constant can be determined between an antibody of the present invention and beta-catenin by SPR at a temperature of 25°C.

[0056] It has been shown that anti-beta-catenin antibodies according to the present invention bind to an epitope located between residues 138 and 390 of intact human beta-catenin (as set forth in SEQ ID NO: 51). Thus, described herein are anti-beta-catenin antibodies that specifically bind to human beta-catenin on an epitope located between residues 138 and 390 of SEQ ID NO: 51. The epitope may be a conformational epitope or a linear epitope.

[0057] Epitopes can be identified by any suitable epitope mapping method known in the art in combination with any one of the antibodies provided by the present invention. An example of such a method involves screening peptides of various lengths derived from full-length beta-catenin for binding to the antibodies of the present invention or fragments thereof, and identifying the smallest fragment capable of specifically binding to the antibody that contains the sequence of the epitope recognized by the antibody. Beta-catenin peptides may be produced synthetically or by proteolytic digestion of beta-catenin. Peptides that bind to antibodies can be identified, for example, by mass spectrometry. The epitope to which an antibody binds can be identified using methods such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, or hydrogen-deuterium exchange mass spectroscopy (HDX-MS). Typically, when determining an epitope by X-ray crystallography, amino acid residues of the antigen within 4 Å of the CDR are considered to be part of the epitope. Once identified, an epitope can be used to prepare fragments that bind to the antibodies of the invention and, if desired, can be used as an immunogen to obtain additional antibodies that bind to the same epitope.

[0058] The epitopes described in the aspects and embodiments of the present invention are preferably epitopes characterized by X-ray crystallography. In one embodiment, the present invention provides an anti-beta-catenin antibody that binds to a conformational or linear epitope on beta-catenin, the conformational or linear epitope comprising at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten conformational or linear epitopes within the region located at residues 164-390.

[0059] In one embodiment of the present invention, for binding to beta-catenin, i. CDR-H1 of SEQ ID NO: 1, 35, or 36; CDR-H2 of SEQ ID NO: 2, 37, or 38; and CDR-H3 of SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii. CDR-H1 of SEQ ID NO: 4; CDR-H2 of SEQ ID NO: 5; and CDR-H3 of SEQ ID NO: 6 or 46; iii. CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; and CDR-H3 of SEQ ID NO: 9, 47, 48, 49, or 50; iv. A sequence according to any one of SEQ ID NOs: 21-25, 28, 31, or 33; v. a sequence according to any one of SEQ ID NOs: 59, 60, 61, or 62; vi. CDR-H1, CDR-H2, and CDR-H3 having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of i-iii); or vii. A sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of iv-v. Provided herein are antibodies that cross-compete with an antibody comprising a heavy chain variable region comprising:

[0060] To determine whether an antibody competes with a reference antibody for binding, the above binding method is performed in two different experimental settings. In the first setting, the reference antibody is allowed to bind to the antigen under saturating conditions, followed by evaluation of the binding of the test antibody to the antigen. In the second setting, the test antibody is allowed to bind to the antigen under saturating conditions, followed by evaluation of the binding of the reference antibody to the protein / peptide. If only the first (saturating) antibody can bind to the protein / peptide in both experimental settings, it is concluded that the test antibody and the reference antibody compete for binding to the antigen. As will be recognized by those skilled in the art, an antibody that competes with a reference antibody for binding does not necessarily have to bind to the same epitope as the reference antibody, but may sterically block the binding of the reference antibody by binding to an overlapping or adjacent epitope, or may undergo a conformational change that results in a lack of binding.

[0061] Two antibodies bind to the same or overlapping epitopes if each competitively inhibits (blocks) the binding of the other antibody to the antigen. Alternatively, two antibodies have the same epitope if mutations of essentially all amino acids in the antigen that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody. Two antibodies have overlapping epitopes if mutations of some amino acids that reduce or eliminate binding of one antibody reduce or eliminate binding of the other antibody.

[0062] Further routine experiments (e.g., peptide mutation and binding analysis) can be performed to confirm whether the observed lack of binding of the test antibody is in fact due to binding to the same part of the antigen as the reference antibody, or whether steric blocking (or another phenomenon) is responsible for the observed lack of binding. These types of experiments can be performed using ELISA, RIA, surface plasmon resonance (SPR), flow cytometry, or any other quantitative or qualitative antibody binding assay available in the art.

[0063] For example, antibodies V1, V2, V3, S, and O have been shown to cross-compete for binding to beta-catenin.

[0064] Those skilled in the art will also understand that antibodies can undergo various post-translational modifications. The type and extent of these modifications often depend on the host cell system and culture conditions used to express the antibody. Such modifications can include variations in glycosylation, methionine oxidation, diketopiperazine formation, aspartate isomerization, and asparagine deamidation. A common modification is the loss of a basic residue (e.g., lysine or arginine) at the carboxy terminus due to the action of carboxypeptidase. Thus, the C-terminal lysine of the antibody heavy chain may be absent. In one embodiment, the C-terminal amino acid from the antibody is cleaved during post-translational modification. In another embodiment, the N-terminal amino acid from the antibody is cleaved during post-translational modification. In certain further embodiments, antibody variants with one or more amino acid substitutions, insertions, and / or deletions are provided. Sites of interest for substitutional mutagenesis include the CDRs and framework regions. Amino acid substitutions are introduced into the antibody of interest, and the products are screened for desired activity, e.g., retention / improvement of antigen binding and / or reduced immunogenicity.

[0065] In certain embodiments, amino acid sequence variants of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and / or other biological properties of the antibody. Amino acid sequence variants of anti-beta-catenin antibodies may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the protein or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the anti-beta-catenin antibody (e.g., one or more CDRs and / or framework sequences in the VH domain). Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired properties.

[0066] In certain embodiments of the variant VH sequences provided herein, each CDR (or HVR) is unaltered or contains only one, two, or three amino acid substitutions.

[0067] It will be appreciated that one or more amino acid substitutions, additions, and / or deletions can be made in the CDRs provided by the present invention without significantly altering the ability of the antibody to bind to and neutralize beta-catenin activity. One skilled in the art can easily test the effect of any amino acid substitution, addition, and / or deletion, for example, by using the methods described herein, particularly those described in the Examples, to determine inhibition of beta-catenin binding and its interaction with its natural interacting partners (e.g., Pontin52, Bcl-9, and lymphoid enhancer factor 1 (LEF-1) / T-cell factor (TCF) family).

[0068] Thus, in certain embodiments of the variant VH sequence, each CDR contains only one, two, or three amino acid substitutions, and such amino acid substitutions are conservative, such that the antibody retains its binding properties to beta-catenin. Thus, provided herein is an antibody that specifically binds to human beta-catenin, the antibody comprising one of the following i to iii: i) CDR-H1 of SEQ ID NO: 1, 35, or 36; CDR-H2 of SEQ ID NO: 2, 37, or 38, and CDR-H3 of SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii) CDR-H1 of SEQ ID NO: 4, CDR-H2 of SEQ ID NO: 5, and CDR-H3 of SEQ ID NO: 6 or 46. H3; iii) a heavy chain variable region comprising CDR-H1, CDR-H2, and CDR-H3 sequences that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of SEQ ID NO: 9, 47, 48, 49, or 50: CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; and CDR-H3 of SEQ ID NO: 9, 47, 48, 49, or 50.

[0069] Antibodies are also provided that further comprise framework regions (FR) comprising or consisting of FR1, FR2, FR3, and FR4 sequences that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of the following i to iv: i. FR1 comprising SEQ ID NO: 10, 15, or 18; ii. FR2 comprising SEQ ID NO: 11, 16, or 19; iii. FR3 comprising SEQ ID NO: 12, 13, 17, or 20; and iv. FR4 comprising SEQ ID NO: 14.

[0070] In alternative embodiments, anti-beta-catenin antibodies of the invention comprise a heavy chain variable region comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in any one of SEQ ID NOs: 21-25, 28, 31, or 33. In further alternative embodiments, anti-beta-catenin antibodies of the invention comprise a heavy chain comprising a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to the sequence given in any one of SEQ ID NOs: 59, 60, 61, or 62.

[0071] The beta-catenin antibody variants provided herein by the present invention retain the advantageous properties of the parent antibody (i.e., the unmodified antibody), i.e., the functional properties described herein. In one example, the anti-beta-catenin antibody variants provided by the present invention have a dissociation constant (K) of 20 nM or less, particularly 15 nM or less, particularly 10 nM or less, for example 9 nM or less, 8 nM or less, 7 nM or less. D In certain embodiments, the kD is as low as 6 nM or less, 5 nM or less, or even 4 nM or less. The KD can be measured / determined by any standard method. For example, the dissociation constant can be determined between an antibody of the invention and beta-catenin by SPR at a temperature of 25°C.

[0072] The degree of identity and similarity between sequences can be readily calculated. "% sequence identity" (or "% sequence similarity") is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) amino acids (e.g., identical amino acids occur in both sequences, similar amino acids occur in both sequences) to obtain the number of matching positions, (3) dividing the number of matching positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to obtain the % sequence identity or percent sequence similarity.

[0073] Methods for aligning sequences for comparison are well known in the art. Preferred examples of algorithms suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms. Polypeptide sequences can also be compared using FASTA, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.

[0074] In certain embodiments, substitutions, insertions, or deletions may be made in one or more CDRs as long as such alterations do not substantially reduce the ability of the antibody to bind to the target.

[0075] For example, conservative changes can be made within the CDRs that do not substantially reduce binding affinity. Such changes can be made outside the antigen-contacting residues within the CDRs.

[0076] Conservative substitutions are shown in Table 1, along with more substantial "exemplary substitutions." [Table 1]

[0077] Substantial alterations in the biological properties of antibody variants can be achieved by choosing substitutions that differ significantly in their effect on maintaining the structure of the polypeptide backbone, the charge or hydrophobicity of the molecule at the target site, or the bulk of the side chains in the substituted areas.

[0078] One type of substitutional variant involves substituting one or more CDR region residues of a parent antibody (humanized or human). Generally, the resulting variant(s) selected for further study will have certain altered biological properties relative to the parent antibody (e.g., increased affinity, decreased immunogenicity) and / or will have certain biological properties of the parent antibody that are substantially retained. An exemplary substitutional variant is an affinity-matured antibody, which can be conveniently generated using affinity maturation techniques, e.g., based on phage display. Briefly, one or more CDR residues are mutated, and the variant antibodies are displayed on phage and screened for a particular biological activity (e.g., binding affinity).

[0079] Alterations (e.g., substitutions) may be made in CDRs, for example, to improve antibody affinity. Such alterations may be made in hypervariable region (HVR) "hot spots," i.e., residues encoded by codons that undergo frequent mutation during the somatic maturation process and / or residues that contact the antigen, and the resulting variant VHs are tested for binding affinity. Affinity maturation by constructing and reselecting secondary libraries is well described in the literature. In some affinity maturation embodiments, diversity is introduced into the variable genes selected for maturation by any of a variety of methods (e.g., error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis). A secondary library is then created. The library is then screened to identify any antibody variants with the desired affinity. One method that can be used to identify antibody residues or regions that can be targeted for mutagenesis is alanine scanning mutagenesis. Alternatively, or in addition, X-ray structures of antigen-antibody complexes can be used to identify contact points between the antibody and its antigen. The variants may be screened to determine whether they contain the desired properties.

[0080] Antibodies raised against beta-catenin may be obtained after immunizing an animal, preferably a non-human animal, by administering beta-catenin or a portion thereof to the animal using well-known and routine protocols. Many animals can be immunized, such as rabbits, mice, rats, sheep, cows, llamas, camels, or pigs.

[0081] Monoclonal antibodies may be produced by a variety of techniques, including, but not limited to, hybridoma methods, recombinant DNA methods, phage display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci. One non-limiting method for producing antibodies is described in the Examples section.

[0082] Also described herein is a method for identifying an antibody according to the invention, said method comprising: a) immunizing a non-human animal with a beta-catenin immunogenic composition; b) recovering B cells from said non-human mammal; c) The following characteristics: i. Dissociation constant K<20 nM D binds to beta-catenin with an affinity expressed as ii. Blocks the binding of any one or more of Pontin52, Bcl-9, and TCF to beta-catenin; and / or iii. Neutralizing or inhibiting the transcriptional activation activity of beta-catenin selecting antibodies produced by said B cells having at least one or more of: Includes.

[0083] The present invention also provides an isolated polynucleotide encoding an antibody according to the invention.

[0084] Isolated polynucleotides according to the present invention can include, for example, synthetic DNA produced by chemical treatment, cDNA, genomic DNA, or mRNA, or any combination thereof.

[0085] The present invention also provides a cloning or expression vector comprising one or more of the polynucleotides described herein. In one example, a cloning or expression vector according to the invention comprises one or more of the isolated polynucleotides described above.

[0086] Standard techniques of molecular biology may be used to prepare DNA sequences encoding the antibodies of the invention. The desired DNA sequence may be synthesized in whole or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as needed.

[0087] General methods for constructing vectors, transfection, and culture methods are well known to those skilled in the art.

[0088] Host cells containing one or more cloning or expression vectors comprising one or more isolated polynucleotide sequences according to the present invention or one or more isolated polynucleotide sequences encoding an antibody of the present invention are also provided. Any suitable host cell / vector system may be used for expressing polynucleotide sequences encoding an antibody of the present invention. Bacteria, such as Escherichia coli (E. coli), and other microbial systems may be used. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable hosts for cloning or expressing antibody-encoding vectors, including strains of fungi and yeast whose glycosylation pathways have been "humanized," resulting in the production of antibodies with partially or fully human glycosylation patterns. Additional host cells that can be used include any eukaryotic cell, e.g., mammalian cells. Suitable mammalian host cells include Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or lymphoid cells (e.g., YO, NS0, Sp20 cells). Suitable CHO cell types according to the invention may include CHO and CHO-K1 cells, including dhfr-CHO cells such as CHO-DG44 and CHO-DXB11 cells used with the DHFR selectable marker, or CHOK1-SV cells used with the glutamine synthetase selectable marker. Other cell types for use in expressing antibodies include lymphoid cell lines, such as NS0 myeloma cells and SP2 cells, COS cells, or human embryonic cells such as HEK293, HEK293F, HEK293S, or EK293T. Host cells may be stably transformed or transfected with the isolated polynucleotide sequence or expression vector according to the invention.

[0089] The present invention also provides a process for producing an antibody according to the invention, comprising culturing a host cell according to the invention under conditions suitable for producing an antibody according to the invention and isolating the antibody.

[0090] The invention also provides a process for producing a pharmaceutical composition comprising an antibody according to the invention, comprising the steps of culturing a host cell according to the invention under conditions suitable for producing an antibody according to the invention, isolating the antibody, and formulating the antibody into a pharmaceutical composition.

[0091] An antibody may comprise only heavy chain polypeptides, in which case only heavy chain polypeptide coding sequences need be used to transfect the host cell (only one vector). To produce antibodies comprising both heavy and light chains, a cell line may be transfected with two vectors: a first vector encoding a light chain polypeptide and a second vector encoding a heavy chain polypeptide. Alternatively, a single vector containing sequences encoding both light and heavy chain polypeptides may be used.

[0092] Thus, provided herein is a process for culturing host cells in accordance with the invention to express an antibody, isolating the antibody, and optionally purifying said antibody to provide an isolated antibody.

[0093] The invention also provides a process for producing an antibody according to the invention, which comprises culturing a host cell comprising a vector of the invention under conditions suitable for expression of protein from DNA encoding the antibody molecule of the invention, and isolating the antibody molecule.

[0094] In one embodiment there is provided an antibody according to the invention which is purified, such as a humanized antibody, particularly in a substantially purified form, particularly free or substantially free from endotoxin and / or host cell proteins or DNA.

[0095] The antibody according to the present invention may be provided in a pharmaceutical composition. Thus, the present invention also provides a medicament comprising an antibody according to the present invention or a polynucleotide encoding the antibody according to the present invention, in combination with one or more pharmaceutically acceptable carriers, excipients, and / or diluents. Preferably, the pharmaceutical composition comprises an antibody that specifically binds to beta-catenin or a polynucleotide encoding such an antibody and one or more pharmaceutically acceptable carriers, excipients, and / or diluents, wherein the antibody is i. CDR-H1 according to SEQ ID NO: 1, 35, or 36; CDR-H2 according to SEQ ID NO: 2, 37, or 38, and CDR-H3 according to SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii. CDR-H1 according to SEQ ID NO: 4, CDR-H2 according to SEQ ID NO: 5, and CDR-H3 according to SEQ ID NO: 6 or 46; iii. CDR-H1 according to SEQ ID NO: 7; CDR-H2 according to SEQ ID NO: 8; and CDR-H3 according to SEQ ID NO: 9, 47, 48, 49, or 50; or iv. A sequence according to any one of SEQ ID NOs: 21-25, 28, 31, or 33; v. a sequence according to any one of SEQ ID NOs: 59, 60, 61, or 62; vi. CDR-H1, CDR-H2, and CDR-H3 having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of i-iii; or vii. A sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of iv-v. The heavy chain variable region comprises:

[0096] The pharmaceutical compositions of the present invention can be used for treatment. The pharmaceutical compositions can be suitably administered to a patient to determine the therapeutically effective amount required. For any antibody, the therapeutically effective amount can be initially estimated using cell culture assays or animal models, usually in rodents, rabbits, dogs, pigs, or primates. Animal models can also be used to determine appropriate concentration ranges and routes of administration. Such information can then be used to determine useful doses and routes of administration in humans.

[0097] The precise therapeutically effective amount for a human subject will depend on the severity of the disease state, the subject's general health, the subject's age, weight, and sex, diet, timing and frequency of administration, drug combination(s), reaction sensitivities and tolerance / response to treatment. Generally, a therapeutically effective amount will be from 0.01 mg / kg to 500 mg / kg, e.g., from 0.1 mg / kg to 200 mg / kg, 100 mg / kg, etc. Pharmaceutical compositions may conveniently be presented in unit dose form, containing a predetermined amount of an active agent of the invention per dose.

[0098] Pharmaceutical compositions according to the invention preferably contain not only an antibody according to the invention or a polynucleotide encoding such an antibody, but also one or more pharmaceutically acceptable carriers, excipients (e.g., buffers, stabilizers, suspending agents, preservatives, and / or dispersing agents, or other materials well known to those skilled in the art) and / or diluents. Such materials should be non-toxic and not interfere with the efficacy of the active ingredient. Examples of carriers, excipients, and / or diluents, as well as methods for preparing pharmaceutical compositions, can be found in Remington's Pharmaceutical Sciences, 20th ed., 2000, pub. Lippincott, Williams & Wilkins.

[0099] Suitable administration forms include forms suitable for parenteral administration, for example, by injection or infusion, e.g., bolus injection or continuous infusion, intravenous, inhalation, or subcutaneous administration. If the product is for injection or infusion, it may be in the form of a suspension, solution, or emulsion in an oily or aqueous vehicle. Alternatively, the antibody according to the present invention may be in a dry form for reconstitution with an appropriate sterile liquid before use. Solid forms suitable for solution or suspension in a liquid vehicle before injection may also be prepared.

[0100] Since the antibodies according to the invention are intrabodies, alternative delivery methods can be used to increase their availability within the cell, such as conjugation to cell-penetrating peptides such as TAT ​​(van den Berg and Dowdy, 2011), engineering the pI of intrabodies to create cell-penetrating antibodies (transbodies) (Lafaye et al., 2011), delivery using viral nanoparticles (Yildiz et al., 2011), or even delivery by carbon nanotubes (Shi Kam et al., 2004).

[0101] Once formulated, the compositions of the invention can be administered directly to the subject or can be reconstituted and then administered according to the appropriate form.

[0102] Thus, there is provided herein the use of an antibody according to the invention or a polynucleotide encoding such an antibody for the manufacture of a medicament.

[0103] Preferably, the pharmaceutical compositions according to the invention are adapted for administration to a primate, such as a human or non-human subject.

[0104] Also encompassed herein is a medicament comprising an antibody that specifically binds to beta-catenin or a polynucleotide encoding such an antibody, and one or more pharmaceutically acceptable carriers, excipients, and / or diluents, for use in therapy, wherein the antibody is i. CDR-H1 of SEQ ID NO: 1, 35, or 36; CDR-H2 of SEQ ID NO: 2, 37, or 38; and CDR-H3 of SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii. CDR-H1 of SEQ ID NO: 4; CDR-H2 of SEQ ID NO: 5; and CDR-H3 of SEQ ID NO: 6 or 46; iii. CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; and CDR-H3 of SEQ ID NO: 9, 47, 48, 49, or 50; iv. A sequence according to any one of SEQ ID NOs: 21-25, 28, 31, or 33; v. a sequence according to any one of SEQ ID NOs: 59, 60, 61, or 62; vi. CDR-H1, CDR-H2, and CDR-H3 having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of i-iii; or vii. A sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of iv-v. The heavy chain variable region comprises:

[0105] Also encompassed herein is an antibody according to the invention, a polynucleotide according to the invention, or a pharmaceutical composition according to the invention for use in therapy, in particular for use in the treatment of the disorders or conditions described herein, wherein such an antibody is administered in a therapeutically effective amount.

[0106] The present invention provides a method of treating a disorder or condition described herein in a subject in need thereof, the method comprising the step of administering to the subject an antibody, polynucleotide, or pharmaceutical composition according to the present invention, wherein such antibody is administered in a therapeutically effective amount.

[0107] The invention also provides the use of an antibody, polynucleotide, or pharmaceutical composition of the invention for the manufacture of a medicament, particularly for use in the treatment of a disorder or condition described herein.

[0108] In one embodiment, the disorder or condition generally related to the present invention is characterized by overexpression of beta-catenin, increased beta-catenin abundance, and / or increased beta-catenin transcriptional activity. Preferably, the disorder or condition is cancer, such as (but not limited to) colorectal cancer, adenomatous polyposis (FAP), colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma, pilomatricoma, and prostate cancer. Alternatively, the disorder or condition generally related to the present invention may be a neurodegenerative disorder, such as Huntington's disease, Alzheimer's disease, Parkinson's disease, prion disease, multiple sclerosis, amyotrophic lateral sclerosis, or schizophrenia, or further disorders, such as bone mineral density disorders, schizophrenia, type II diabetes, rheumatoid arthritis, oligodontia, osteoporosis-pseudoglioma syndrome, familial exudative vitreoretinopathy, or even vascular calcification.

[0109] In alternative embodiments, there is also provided herein an antibody according to the invention, a polynucleotide according to the invention, or a pharmaceutical composition according to the invention for use in preventing excessive growth of polyps in FAP patients. [Brief explanation of the drawings]

[0110] [Figure 1] Screening of intracellular function of VHH intrabodies transiently transfected into HEK293 bioassay cells. Wnt signaling was induced by co-transfection of the PPSP gene. A)C)E)G)I): ​​Wnt-induced firefly luciferase activity. Results plotted as a percentage of cells transfected with a control VHH (CAb). B)D)F)H)J): Constitutive Renilla luciferase activity. Results plotted as a percentage of cells transfected with a control VHH (CAb). Asterisks indicate the VHH intrabody of interest. Each set of graphs represents a different plate. Error bars indicate SEM. [Figure 2] Secondary screening of intracellular function of VHH intrabodies transiently transfected into HEK293 bioassay cells. Wnt signaling was induced by co-transfection of the Wnt1 gene. A)C): Firefly luciferase activity. Results are plotted as fold stimulation over vector-transfected inactivated cells. All conditions were performed in triplicate. B)D): Renilla luciferase activity. Results are plotted as raw signal. Asterisks indicate previously selected intrabodies of interest. Error bars indicate SEM. [Figure 3] Sequence alignment of functionally engineered intrabody VHH sequences that showed functional activity against beta-catenin transcriptional activity in a HEK293 luciferase-based bioassay. The three CDRs are highlighted in bold and underlined font. [Figure 4] Alignment of the parental VHH and the designed CDR3 mutants. [Figure 5] Reduced anti-beta-catenin Western blots probed with intrabody: Gel A: IBV1-scFc (23 nM), Gel B: IBV2-scFc (110 nM), Gel C: IBV3-scFc (85 nM), and Gel D: IBV1.CDRAAA-scFc (16 nM) transient supernatants. Binding was detected with anti-mouse Fc HRP antibody. In each gel, lane 1: 15 ng of beta-catenin, lane 2: 7.5 ng of beta-catenin, and lane 3: 3.75 ng of beta-catenin. [Figure 6] β-catenin binding ELISA results for parental VHH and CDR3 variants. Biotinylated β-catenin was captured on streptavidin-coated plates, and VHHscFc constructs were then added at half-log dilutions starting from 32 nM and developed with anti-mouse Fc HRP. Plates were read at 630 nm and 490 nm, and OD was recorded. The negative control was a "no DNA" control transfection culture supernatant. [Figure 7]Activity of parental intrabodies and CDR3 mutant control intrabodies in bioassays. Pairs of parental intrabodies and selected CDR3 mutant control intrabodies, IBV1 and IBV1.CDRAAA, IBV2 and IBV2.CDRAAA, and IBV3 and IBV3.CDRAAA (1), were tested for firefly and Renilla luciferase activity in bioassays. "A" and "B": Firefly luciferase activity. Results are plotted as fold stimulation over transfected but inactivated cells. All conditions were performed in quintuplicate and represent four independent experiments. "C" and "D": Renilla luciferase activity. Results are plotted as raw data. For two-tailed unpaired Student's t-test, **** indicates p ≤ 0.0001, *** indicates p 0.0001–0.001, ** indicates p 0.001–0.01, and * indicates p 0.01–0.05. Error bars indicate SEM.

[0111] Array Description [Table A-1] [Table A-2] [Table A-3] [Table A-4] [Table A-5] [Example]

[0112] method Production of recombinant beta-catenin protein: A full-length human beta-catenin cDNA clone was obtained from Origene. Beta-catenin was expressed and purified essentially as described (Xing Y et al., 2008).

[0113] Generation of immune VHH phage library: Two male llamas were immunized with five doses of 0.25 mg of recombinant beta-catenin in Freund's adjuvant over a period of three months. PBMCs were collected from these animals at different time points (bleeds), and RNA was purified using the RNeasy kit (Qiagen). VH genes were amplified by PCR following RT of purified RNA with an oligo-dT primer using primers "llimmlibfor" (SEQ ID NO: 52) and "CH2Ig primer" 54 (SEQ ID NO: 53). VHH genes were specifically identified from the resulting PCR products based on size, reamplified using specific primers, and cloned into pASTT. The ligated products were electroporated into XL-1 Blue E. coli (Stratagene), and the library was grown at 2 × 10 8 Phage particles were prepared using standard methods.

[0114] Phage panning: In the first panning experiment, the immune library was incubated in beta-catenin-conjugated immunotubes blocked with 3% bovine serum albumin (BSA). Two distinct wash protocols were then performed: a low wash protocol (5 washes in round 1 and 10 washes in round 2) and a high wash protocol (20 washes in round 1 and 40 washes in round 2). The phage were then eluted with 0.1 M HCl and subsequently neutralized. In the second round of panning, input phage from both wash strategies were panned against both beta-catenin-conjugated and unconjugated immunotubes. One round of panning was also completed with the antigen in solution. Recombinant biotinylated beta-catenin was incubated with 6 × 10 β-catenin-conjugated immunotubes preblocked with phosphate-buffered saline (PBS) containing 2.5% milk. 11The beads were incubated (spin) with library phage particles. Beta-catenin and associated phage were then captured from solution using M280 streptavidin-conjugated Dynabeads® (Invitrogen). The beads were washed four times by magnetic capture, and bound phage were eluted as described above. Small-scale phage rescue was performed on individual E. coli colonies picked from output phage-infected colonies from each round of panning conditions. Each sample was tested for binding to beta-catenin or streptavidin by ELISA. Samples with antigen-binding signals greater than three times the negative signal were determined to be positive "hits."

[0115] Bioassay screening: HEK293 cells stably transfected with the Tcf-firefly luciferase reporter construct according to standard protocols were grown in DMEM (Gibco Life Technologies) supplemented with 10% fetal bovine serum, 2 mM glutamine, and non-essential amino acids (Gibco Life Technologies). 5 × 10 cells were plated onto poly-D-lysine-coated white 96-well plates. 4 Cells were seeded at 1 / well and transfected with an expression plasmid (containing 10 ng of Promega's Renilla luciferase pGL4.74 plasmid) using Lipofectamine 2000 (Gibco Life Technologies) according to the manufacturer's instructions. Plates were incubated at 37°C in 5% CO for 40 hours. Where necessary, Wnt3a-conditioned medium or LiCl was added 18 hours after transfection. After incubation, firefly and Renilla luciferase activities were measured using Dual-Glo® (Promega) according to the manufacturer's instructions. Luminescence was recorded using a Biotek Synergy plate reader.

[0116] ELISA: ELISA plates were coated overnight at 4°C. Washing was performed between each step of the assay and consisted of four washes in PBS (containing 0.1% Tween 20). All plates were blocked with 3% BSA in PBS, and all samples were blocked with 2.5% milk in PBS for at least 1 hour before adding screening samples to the ELISA plates. Following the final wash, TMB (Calbiochem) was added, and the OD of the plates was read at 630 nm and 490 nm using a Biotek Synergy plate reader, and the delta OD (ΔOD) was recorded.

[0117] Beta-catenin binding ELISA: ELISA plates were coated with streptavidin (2 mg / mL). Biotinylated beta-catenin (1 mg / mL) was added in PBS for 30 minutes. Blocked samples were added (phage samples or transient VHHscFc samples). After 1 hour, bound samples were detected with the appropriate antibody at a 1:5000 dilution in PBS (containing 3% BSA). The detection antibodies were goat anti-M13-HRP (GE Healthcare) or goat anti-mouse Fc HRP (Jackson ImmunoResearch).

[0118] Streptavidin-binding ELISA: ELISA plates were coated with streptavidin (2 mg / mL). Blocked samples were added. After 1 hour, bound samples were detected with the appropriate antibody at a 1:5000 dilution in PBS (+3% BSA).

[0119] Confocal microscopy: HEK293 cells transfected with Myc-His tagged VHH constructs were attached to poly-D-lysine-coated 8-well culture microscope slides in complete medium. Cells were fixed with 4% paraformaldehyde and blocked / permeabilized in TBS block (0.3% Triton 100, 1% BSA, and 5% goat serum) for 1 hour. Anti-myc (9E10) Alexa Fluor® 488 (Gentaur) and anti-beta-catenin (6B3) (Cell Signaling Technologies) were then added and incubated overnight at 4°C in a humidified chamber. An anti-beta-catenin detection antibody, anti-rabbit Alexa Fluor® 647 (Jackson ImmunoResearch), was added at a 1:1000 dilution and incubated in the dark for 1 hour. Finally, DAPI ProLong® Gold Antifade (Invitrogen) was added along with a coverslip. Images were captured using a Leica TCS SP5 microscope.

[0120] Production of VHH-scFc protein: HEK293 cells were transfected with the VHH-scFc expression vector using 293fectin™ (Invitrogen) according to the manufacturer's instructions. The single-chain mouse IgG Fc construct consisted of a VHH, a hinge, the CH2 and CH3 domains connected to another hinge, and the CH2 and CH3 sequences via a 59-amino acid linker (SEQ ID NO: 54). This construct has previously been demonstrated as a tag for VHH (data not shown), which aids in extracellular expression, purification, and oriented immobilization. Transfected HEK293 cells were incubated at 37°C in 5% CO2 on a shaking platform for 5-6 days. Cells were then harvested from the culture by centrifugation at 423 × g, and the supernatant containing VHH-scFc was stored at 4°C.

[0121] Immunoprecipitation: 2 x 10 6HEK293 bioassay cells were washed with ice-cold PBS and resuspended in 200 mL of ice-cold cell lysis buffer (containing a protease inhibitor cocktail). The samples were then rotated at 4°C for 30 minutes, followed by centrifugation at 10,000 × g for 15 minutes at 4°C. The HEK293 cell lysate supernatant was then transferred to a new chilled microcentrifuge tube. VHH-scFc was conjugated to sheep anti-mouse Fc Dynabeads® (Invitrogen) according to the manufacturer's instructions. The beads were then added to 500 mL of HEK293 cell lysate and rotated overnight at 4°C. Each set of beads was magnetically captured from the solution and washed three times individually with fresh cell lysis buffer. Protein was then eluted from the beads by boiling for 5 minutes in LDS sample buffer containing dithiothreitol (DTT) (Invitrogen). Samples were then analyzed by anti-beta-catenin and anti-VHH-scFc Western blot.

[0122] Protein SDS-PAGE and immunological detection (Western blot): Appropriate amounts of protein samples were separated by SDS-PAGE on 4-12% Bis-Tris gels (Invitrogen) and electroblotted onto PVDF membranes using iBLOT (Invitrogen). The membranes were probed by incubation with the appropriate antibodies. These included mouse anti-myc (9E10, Gentaur) and goat anti-mouse Fc HRP (Jackson ImmunoResearch) for the detection of VHHs with and without scFc tags; biotinylated "O" (produced in-house), rabbit anti-beta-catenin (6B3, Cell Signaling Technologies), and rabbit anti-penta-HIS (Bethyl Laboratories), respectively, followed by streptavidin-HRP (Jackson ImmunoResearch) or anti-rabbit Fc HRP (Jackson ImmunoResearch). All membranes were developed with Pico Super Signal ECL (Pierce) for 5 minutes, images were captured using an ImageQuant LAS 4000 (GE Healthcare), and the resulting band densitometry was analyzed using ImageQuant TL analysis software.

[0123] Generation of beta-catenin fragments: BL21 Star™ DE3 cells (Invitrogen) expressing each of the beta-catenin constructs were grown overnight with shaking in antibiotic-selective 2xTY medium supplemented with IPTG (final concentration 0.3 mM). The overnight cultured cells were harvested by centrifugation at 4,000 x g for 5 minutes. Pelleted cells were lysed by rotating at room temperature for 20 minutes with 1x Bugbuster® (Novagen) containing Lysonase (Novagen) in 20 mM sodium phosphate, 500 mM NaCl, 20 mM imidazole, and 2 mM DTT.

[0124] SPR-Affinity Measurements: Affinity measurements were performed using a Biacore™ T100. Streptavidin was immobilized on the carboxymethylated dextran-coated gold surface of a CM5 sensor chip using amine coupling chemistry according to the manufacturer's instructions. The streptavidin concentrations used in flow cells 1–4 were 0 μg / mL, 5 μg / mL, 15 μg / mL, and 45 μg / mL, respectively, in 10 mM NaOAc at pH 5. Biotinylated beta-catenin was then captured on flow cells 2–4, with flow cell 1 serving as a control reference cell. Samples of VHH-scFc diluted 2x, 5x, 10x, 20x, and 40x in HBS-EP were passed through all flow cells at 30 μL / min for 5 min. A buffer blank was run between each sample set, and a control transfection supernatant sample was included in each run. Between each sample, the chip was regenerated by adding 30 μL of 2 M guanidine hydrochloride followed by 30 μL of 40 mM HCl. Affinity constants were determined by fitting the resulting data to a Langmuir 1:1 association law using BiaEvaluation software. Associations were rejected if the chi-squared value was greater than 1, if the remaining data points were not randomly distributed, or if the residuals were greater than ±2 RU. Association data were plotted for the most diluted sample that still gave an RU capture response of at least 40 RU. The minimum values ​​among the four different dilution combinations and different beta-catenin-coated flow cells were used to calculate the "on-rate" (ka) of association or the "off-rate" (kd) of dissociation data, which were used to calculate KD = kd / ka.

[0125] (Example 1) Generation of a VHH anti-beta-catenin immune phage display library Two llamas were immunized as described above. After the fifth dose, serum titers did not further improve, so immunizations were discontinued. Serum response ELISA indicated the production of beta-catenin-binding antibodies, but did not provide information on the isotype of the antibodies produced, as anti-llama IgG antibodies recognized both conventional and HCAb isotypes. Using standard protocols, antibody isotypes with specificity for beta-catenin were identified by immunoprecipitation with beta-catenin-coated beads and compared with the amount and isotype of antibodies precipitated with uncoated beads as a control. The results indicated that enrichment occurred when beta-catenin was present, capturing greater amounts of conventional and heavy chain-only antibodies. Immunoprecipitation experiments with llama serum demonstrated that the two llamas had an immune response to beta-catenin, including HCAb.

[0126] Immune llama phage libraries were then generated (as described in Materials and Methods above). Following on from the monoclonal phage rescue, large-scale polyclonal phage rescue was completed for the entire library, and the sequences were further classified into subfamilies based on the criteria of Harmsen (Harmsen et al., 2000) (see Table 2 below). Both conventional IgG and HCAbs capable of binding to beta-catenin were identified in the post-immunization serum, identifying members of each of the VHH subfamilies 1-3 previously found in llamas. [Table 2]

[0127] (Example 2) Identification of intrabody variable regions Selection of antigen-specific antibodies from a phage library typically involves repeated rounds of a process commonly known as panning. Multiple rounds of the panning process are usually necessary due to the inefficiency of removing all non-specific antigen-binding phage particles during panning.

[0128] The goal of this study was not to identify any specific anti-beta-catenin antibodies but to select a diverse array of specific anti-beta-catenin VHHs that bind to intracellular beta-catenin in its native intracellular conformation. However, such selection would have been technically challenging using conventional phage display. Therefore, due to practical considerations, we used recombinant beta-catenin displayed both in immunotubes and in solution to maximize the number of available epitopes. Each of these panning conditions was extended to better mimic the intracellular environment, for example, by adding reducing agents.

[0129] To identify individual antibody variable regions with specificity for beta-catenin, a total of 855 small-scale phage rescues and antibody pIII elicitation were performed on individual colonies in a 96-well block format. Using these methods, samples in both rescued antibody-phage fusion and elicited antibody pIII formats were screened for binding. Furthermore, each sample in both formats was tested for binding to beta-catenin captured on an ELISA plate and a streptavidin-coated plate to determine whether the recorded signal was due to binding to beta-catenin. Phage rescue samples were also analyzed for phage-VHH production. From this study, 73 sequences with promising binding to beta-catenin were identified. After cloning into a mammalian expression vector, the antibodies were expressed intracellularly and tested for their ability to modify beta-catenin function. A total of 61 of the 73 sequences were successfully cloned.

[0130] To evaluate the anti-beta-catenin neutralizing activity of these antibodies, we performed a Wnt signaling activity assay using HEK293 cells stably transfected with a reporter construct containing 16xTCF / LEF binding sites upstream of a firefly luciferase reporter gene. The intrabodies were expressed in the cells by transfection with vector DNA, and inhibition of beta-catenin co-transcriptional activity was detected by a decrease in the amount of luciferase produced. This assay enabled the direct identification of intrabodies that modulate canonical Wnt signaling.

[0131] Subcloned VHH intrabodies were transfected into HEK293 bioassay cells at 100 ng / well in triplicate. Firefly luciferase expression was stimulated by cotransfection with 50 ng per well of DNA encoding the LGR6 PPSP motif A peptide (Tamai et al., 2004). After 40 hours of incubation at 37°C, cells were lysed, and firefly and Renilla luciferase activities were measured independently. All plates were transfected in duplicate, allowing the levels of expressed VHHs to be visualized by Western blot using a single plate. Sixty-one VHH intrabodies were analyzed in this manner, and the results are shown in Figure 1.

[0132] Intrabodies were screened for their intracellular function in response to beta-catenin co-transcriptional activity in a HEK293 bioassay. To facilitate comparison of samples across plates, all luciferase activity signals were plotted as a percentage of the control VHH transfected on each screening plate (CAb signal). Some sequences clearly affected the individual luciferase activity signals; for example, intrabodies 6, Z1-3, and R all significantly reduced Renilla luciferase activity and exhibited cytotoxicity. Intrabodies X1 and X2 both showed some stimulation of firefly luciferase activity over the CAb signal, and intrabodies V1-3 all exhibited strong inhibition of firefly luciferase signal. Furthermore, the most diverse family based on sequence was the largest intrabody family, W, which also produced the most diverse luciferase activity signals based on bioassay results. From the 61 intrabodies tested, seven VHHs (all V intrabody families V1, V2, and V3, as well as intrabodies R, S, O, and 15) were selected as intrabodies for further analysis. These intrabodies were selected because they all showed very strong inhibition of firefly luciferase activity signals, with the stimulated luciferase signal recorded by the control VHHs being 25% or less (see Figures 1A, C, E, G, and I). Intrabodies V2, V3, O, and 15 also all maintained Renilla luciferase activity signals similar to those recorded by the CAbs, indicating that the inhibition of firefly luciferase activity was specific (see Figures 1B, D, F, H, and J). Intrabodies V1 and S did show some inhibition of Renilla luciferase activity, but this was not as strong as the inhibition of firefly luciferase activity and was never less than the signal seen for Renilla luciferase activity of the VHH-free mammalian expression vector control included on each plate. Intrabody R did show greater than 50% inhibition of the Renilla luciferase signal, indicating that its inhibitory activity on firefly luciferase activity may not be specific.All seven intrabodies were carried forward to the next stage of screening.

[0133] These intrabodies (selected from the primary screen and shown to be expressed by Western blot; data not shown) were then rescreened in a bioassay using a different stimulus, i.e., WNT1-transfected DNA. Two beta-catenin-binding intrabodies (W3 and C) that did not previously demonstrate inhibition in the bioassay but did bind to beta-catenin were included on each plate as "negative" controls, and inhibitory intrabodies R, S, and V1 were included on each plate as "positive" controls. Additionally, to test whether VHHs generally behave similarly under both bioassay stimuli, various other intrabodies that had not previously affected the bioassay were included across the two screening plates.

[0134] The results of the WNT1-stimulated bioassay (Figure 2) again showed that intrabodies V1, V2, V3, S, and O (also designated herein as IBV1, IBV2, IBV3, IBS, and IBO) demonstrated inhibition of firefly luciferase activity compared to all other intrabodies transfected on the respective plates. IBV1, IBV2, IBV3, and IBO also demonstrated no inhibition of Renilla luciferase activity. Intrabody S again demonstrated some inhibition of Renilla luciferase activity, but not to the same extent as the inhibition of firefly luciferase activity. Intrabodies R and 15 did not appear to be able to inhibit WNT1-induced firefly luciferase activity beyond the inhibition they exhibited against constitutively expressed Renilla luciferase activity. This suggests that their activity is not specific to beta-catenin-mediated inhibition of firefly luciferase production, and therefore they were not investigated further. The sequences of the variable regions of five intrabodies capable of inhibiting beta-catenin-mediated firefly luciferase production are shown in Figure 3. These intrabodies belong to three families based on the variability of their CDR3s.

[0135] (Example 3) Characterization of functionally modified intrabodies To confirm that intrabody activity is dependent on antigen binding, five of the more promising antibodies identified in Example 2 (parental) were mutated in their CDR3s in an attempt to block beta-catenin binding with minimal perturbation of other parts of the VHH domain. These mutations were selected by carefully examining the CDR3 for important binding residues, such as tyrosine, based on the literature. In addition, complete CDR3 grafts were also designed for each parental antibody. Specific CDR3s to graft were identified by aligning all previously identified available sequences against the parental intrabody sequences, and CDR3s of similar length were selected from the most homologous antibodies (Figure 4).

[0136] Reduced beta-catenin samples were electrophoresed on four 4-12% Bis-Tris gels and transferred to PVDF membranes. The membranes were probed with IBV1, IBS, and IBO, all in VHH-scFc format, and IBV1.CDRAAA as a control, and revealed with goat anti-mouse HRP (Figure 5). The presence of beta-catenin was successfully detected in all three parental VHHs but not in the control variant IBV1.CDRAAA. These data strongly suggested that all three parental VHHs bind to linear epitopes of beta-catenin rather than conformational epitopes.

[0137] The binding of the parental intrabodies and CDR3 mutants to recombinant β-catenin was also tested by ELISA. All of these showed binding to β-catenin (Figure 6). Among the various CDR3 mutants, only V1.CDRA, V1.CDRAA, and O.CDRAAA (2) showed significant binding to β-catenin. O.CDRAAA (1) demonstrated all of the desired characteristics of the control VHH for the parental intrabody. IBV1.CDRAAA and IBS.CDRAAA also appeared to be suitable CDR3 mutant controls for their respective parents, except for their low expression levels in bioassays. This is not uncommon in conventional antibody expression; single-point mutations in antibody CDRs have previously been reported to reduce expression by more than 100-fold.

[0138] Neither intrabody O nor intrabody O.CDRAAA(1) had an inhibitory effect on Renilla luciferase activity in bioassays, suggesting that expression of these VHHs was not detrimental to cell viability. The reduced Renilla luciferase production for IBV1.CDRAAA and IBS.CDRAAA compared to IBV1 and IBS indicated that intracellular expression of these CDR3 variants may be sufficient to reduce cell viability. This again demonstrates the different effects that expression of distinct VHH sequences can have on mammalian cells.

[0139] Upon repeated testing, parental intrabodies V, S, and O consistently demonstrated statistically significant inhibition of PPSP- and Wnt1-induced signaling compared with these selected CDR3 mutant control intrabodies. Inhibition of luciferase activity by the parental intrabodies was specific to beta-catenin-induced firefly luciferase production, not specific to Renilla luciferase activity (see Figure 7). While some statistical significance was noted for the differences in Renilla luciferase activity between the parental intrabodies and their respective CDR3 mutant control intrabodies, this was generally due to inhibition by the CDR3 mutant control intrabodies rather than the parental intrabodies. Thus, the parental intrabodies had greater Renilla luciferase activity than the CDR3 mutant control intrabodies. When the data were replotted to account for the relative changes in Renilla luciferase activity, the percentage inhibition of intrabodies V1 and S increased by an average of 10%. These data confirmed that the inhibition of firefly luciferase production by intrabodies V, S, and O was the result of their ability to modulate beta-catenin function and not the result of off-target effects caused by expression of the VHH domains in cells, demonstrating the successful generation of functionally modified intrabodies to the Wnt signaling pathway.

[0140] It was interesting to note that several of the engineered CDR3 mutants retained specific binding to beta-catenin. Mutants IBV1.CDRA and IBO.CDRAAA (2) retained binding to beta-catenin, as detected by ELISA and SPR. They also demonstrated specific inhibition of firefly luciferase activity in bioassays. IBO.CDRAAA (2) retained much of the bioassay activity of IBO despite three Ala substitutions in CDR3. The Ala substitutions involved a pair of bulky and charged arginine residues that may have altered the structure and binding capacity of CDR3. Clearly, this was not the case (as supported by the literature), suggesting that arginines in CDR3 generally do not contribute to affinity for antigens or necessarily correlate with the specificity of antibody-antigen interactions.

[0141] The off-rate data for VHH binding to beta-catenin appeared to correlate well with the activity of the VHH in bioassays. For example, IBV1.CDRA and IBO.CDRAAA (2) had the best off-rates and the greatest activity in bioassays among all variants (data not shown). This is most likely a result of the high intracellular concentrations of beta-catenin or intrabody, implying that regardless of the on-rate, the intrabody binds to beta-catenin, and the determining factor for activity is how long it remains bound. The slow on-rate may be the reason why more binding was recorded by ELISA than by SPR for some CDR3 variants, such as IBV1.CDRAA. Affinity values ​​for VHH families V, S, and O were determined by SPR and ELISA. KD values ​​determined by SPR can be found in Table 3 below. [Table 3]

[0142] All KD values ​​obtained by ELISA revealed affinity values ​​below 10 nM. Recorded affinity values ​​obtained by SPR indicated that the best affinities were obtained for S and O. For intrabodies from family V, SPR demonstrated slightly better affinity for V2 and V3 compared to V1. A single amino acid change between IBV1, IBV2, and IBV3 appeared to have a small effect on their relative affinities. Both IBV2 and IBV3 contain an additional isoleucine rather than threonine or methionine in CDR3, respectively, and both have slightly greater affinity for beta-catenin than IBV1.

[0143] SPR studies showed that IBV1, IBV2, IBV3, IBO, and IBS all "cross-block" each other, i.e., an intrabody could not bind to beta-catenin if another intrabody was already bound.

[0144] In summary, three intrabody families were discovered that specifically affected beta-catenin co-transcriptional activity through CDR3-mediated interactions. Each of the parental VHHs appeared to "cross-block" each other. The most suitable intrabody and CDR mutant intrabody control pair was intrabody IBO and IBO.CDRAAA(1), which also demonstrated the highest percentage of inhibition of firefly luciferase production (83%) in bioassays. Therefore, intrabodies IBO and IBO.CDRAAA(1) were taken forward for further study.

[0145] (Example 4) Mode of action As shown in previous examples, the anti-beta-catenin VHHs of the present invention consistently demonstrated inhibition of the Wnt signaling pathway as measured by inhibition of firefly luciferase production stimulated by Wnt1 or PPSP in a HEK293 bioassay.

[0146] To better understand the mode of action of the anti-beta-catenin antibodies identified herein, we further studied one of them (antibody IBO). We also investigated the use of two additional stimuli, Wnt3a-conditioned medium and LiCl2, as well as alternative bioassays. Both Wnt3a and Wnt1 signal in a similar canonical manner, but are thought to use different sets of frizzled coreceptors. LiCl2 is a potent inhibitor of GSK-3β, preventing it from phosphorylating beta-catenin. Compared to the CDR3 control mutant intrabody IBO.CDRAAA(1), IBO demonstrated 66% inhibition of beta-catenin co-transcriptional activity in HEK293 bioassays stimulated with Wnt3a-conditioned medium and 65% inhibition when stimulated with LiCl2. The fact that IBO could still inhibit firefly luciferase signaling indicates that the intrabody IBO-beta-catenin interaction is downstream of the GSK-3β-beta-catenin interaction. Using a series of truncated beta-catenin fragments and Western blot analysis, IBO was shown to bind to beta-catenin between residues 138 and 390, a region that coincides with the binding domains of Pontin52, Bcl-9, and TCF to beta-catenin.

[0147] Importantly, IBO has been shown to bind to endogenous beta-catenin, and the binding site has been demonstrated to reside between residues 138 and 390. Initial indications were that intrabody IBO specifically disrupts the co-transcriptional activity of beta-catenin without affecting its role at the plasma membrane (data not shown).

[0148] Overall conclusion In summary, the present invention describes novel VHH-based antibodies and active fragments thereof that are capable of disrupting the biological activity of b-catenin in cells, and therefore can be used as intrabodies to treat disorders involving beta-catenin or the beta-catenin pathway, such as those generally described herein. (References) 1) Biocca et al., 1995, Biotechnology, 13:1110-1115 2) Stocks et al., 2005, Curr. Opin. Chem. Biol., 9:359-365 3) Serruys et al., 2009, Hepatology, 49:39-49 4) Vercruysse et al., 2010, J. Biol. Chem., 285:21768-21780 5) Ben-Ze’ev and Geiger, 1998, Curr. Opin. Cell Biol., 10:629-639 6) Morin et al., 1999, BioEssays, 21:1021-1030 7) Newnham et al., 2015, mAbs, 7(1):180--191; 8) Miyaoka et al., 1999, Schizophr. Res., 38 :1-6. 9) Grant et al., 2006, Nat. Genet., 38:320-323 10) Sen et al., 2000, PNAS, 97:2791-2796 11) Lammi et al., 2004, Am. J. Hum. Genet., 74:1043-1050 12) Gong et al., 2001, Cell, 107:513-523 13) Toomes et al., 2004, Am. J. Hum. Genet., 74:721-730. 14) Bienz and Clevers, 2000, Cell,103:311-320 15) McCrea et al., 1993, J. Cell Biol., 123:477-484 16) WO2021015419 17) Kabat et al., 1991, Sequences of Proteins of Immunological Interest, NIH publication No. 91-3242 18) Chothia, C. and Lesk, A.M., 1987, J. Mol. Biol., 196:901-917 19) Angal et al., 1993, Mol Immunol, 30:105-108 20) Kashmiri et al., 2005, Methods, 36, 25-34 21) van den Berg and Dowdy, 2011, Curr. Opin. Biotechnol., 22:888-893 22) Lafaye et al., 2011 (US2011250211) 23) Yildiz et al., 2011, Curr. Opin. Biotechnol., 22:901-908 24) Shi Kam et al., 2004, J. Am. Chem. Soc., 126:6850-6851 25) Xing Y et al., 2008, Structure, 16:478-487 26) Harmsen et al., 2000, Mol. Immunol., 37:579-590 27) Tamai et al., 2004, Mol. Cell, 13:149-156

Claims

1. An antibody that specifically binds to beta-catenin, i. CDR-H1 of SEQ ID NO: 1, 35, or 36; CDR-H2 of SEQ ID NO: 2, 37, or 38, and CDR-H3 of SEQ ID NO: 3, 39, 40, 41, 42, 43, 44, or 45; ii. CDR-H1 of SEQ ID NO: 4, CDR-H2 of SEQ ID NO: 5, and CDR-H3 of SEQ ID NO: 6, 44, 45, or 46; iii. CDR-H1 of SEQ ID NO: 7; CDR-H2 of SEQ ID NO: 8; and CDR-H3 of SEQ ID NO: 9, 47, 48, 49, or 50; or iv) CDR-H1, CDR-H2, and CDR-H3 sequences that have at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity or similarity to any of the sequences listed in any one of i-iii. The above antibody, comprising a heavy chain variable region comprising:

2. i. FR1 comprising SEQ ID NO: 10, 15, or 18, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; ii. FR2 comprising SEQ ID NO: 11, 16, or 19, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; iii. FR3 comprising SEQ ID NO: 12, 13, 17, or 20, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto; and iv. FR4 comprising SEQ ID NO: 14 or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto. The antibody of claim 1, further comprising a framework region (FR) comprising or consisting of:

3. 3. The antibody of claim 1 or 2, having a heavy chain variable region comprising any one of SEQ ID NOs: 21, 22, 23, 24, 25, 28, 31, or 33, or an array having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

4. 4. The antibody of claim 1, which is a VHH or Hcab.

5. The antibody of any one of claims 1 to 4, which is an intrabody.

6. 6. The antibody of any one of claims 1 to 5, which is chimeric or humanized.

7. 7. The antibody of any one of claims 1 to 6, having a heavy chain comprising any one of SEQ ID NOs: 59, 60, 61, or 62, or a sequence having at least 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity thereto.

8. 8. The antibody of any one of claims 1 to 7, which i) blocks the binding of beta-catenin to any one or more of Pontin52, Bcl-9, and lymphoid enhancer factor 1 (LEF-1) / T-cell factor (TCF) family (TCF), and / or ii) neutralizes or inhibits a pathway mediated by beta-catenin.

9. 9. The antibody of claim 1, having an equilibrium dissociation constant (KD) for beta-catenin of less than 20 nM.

10. 10. The antibody of any one of claims 1 to 9, which specifically binds to human beta-catenin on an epitope located between residues 138 and 390 of SEQ ID NO:

51.

11. An antibody that cross-competes with the antibody of any one of claims 1 to 10 for binding to beta-catenin.

12. An isolated polynucleotide encoding the antibody of any one of claims 1 to 11.

13. A cloning or expression vector comprising the polynucleotide of claim 12.

14. A host cell comprising the polynucleotide of claim 12 or the expression vector of claim 13.

15. 15. A process for producing an antibody according to any one of claims 1 to 11, comprising culturing a host cell according to claim 14 under conditions suitable for producing the antibody and isolating the antibody.

16. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 11 or a polynucleotide according to claim 12 and one or more pharmaceutically acceptable carriers, excipients and / or diluents.

17. 17. An antibody according to any one of claims 1 to 11, a polynucleotide according to claim 12, or a pharmaceutical composition according to claim 16, for use in therapy.

18. An antibody according to any one of claims 1 to 11, a polynucleotide according to claim 12, or a pharmaceutical composition according to claim 16, for use in the treatment of a disease or condition characterized by overexpression of beta-catenin.

19. 19. The antibody of any one of claims 1 to 11 and 17 to 18, the polynucleotide of claim 12, 17, or 18, or the pharmaceutical composition of claims 16 to 18, for use in the treatment of any one of (i) cancers such as colorectal cancer, adenomatous polyposis (FAP), colon cancer, melanoma, hepatocellular carcinoma, ovarian cancer, endometrial cancer, medulloblastoma, pilomatricoma, and prostate cancer; (ii) neurodegenerative disorders such as Huntington's disease, Alzheimer's disease, Parkinson's disease, prion diseases, multiple sclerosis, amyotrophic lateral sclerosis, or schizophrenia; or (iii) bone mineral density disorders, schizophrenia, type II diabetes, rheumatoid arthritis, oligodontia, osteoporosis-pseudoglioma syndrome, familial exudative vitreoretinopathy, or even vascular calcification.

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