MAGEB2-binding constructs

Abstract

The present invention relates to a binding construct comprising a domain which binds to MAGEB2. The present invention further provides polynucleotides encoding the binding construct, vectors comprising said polynucleotides and host cells transformed or transfected with said polynucleotides or vectors. The present invention further provides processes for producing the binding construct, therapeutic methods using the binding construct, diagnostic uses of the binding construct and kits comprising the binding construct.

Description

[Technical field]

[0001] The field of the invention relates to compositions and methods relating to the treatment and diagnosis of cancer, including binding constructs that bind to MAGEB2. [Background technology]

[0002] The MAGE (melanoma antigen gene) family includes approximately 60 genes classified into several subfamilies. The MAGE-A, -B and -C subfamilies are primarily expressed in the testis and are aberrantly expressed in various cancer types. The MAGE-D, -E, -F, -G, -H, -L and -N subfamilies are expressed in diverse tissues. See, for example, Lee and Potts, J. Mol. Biol., 2018. One MAGE family member, MAGEB2, is typically expressed only in normal testis. MAGEB2, which may function to enhance the ubiquitin ligase activity of RING-type zinc finger-containing E3 ubiquitin protein ligases, has been found to be aberrantly expressed in various human tumors, such as lung cancer, breast cancer and melanoma. Given this aberrant expression, MAGEB2 is a potential target for new therapeutic agents.

[0003] Early detection and classification of cancer is a key factor for successful treatment of the disease. A sensitive and accurate diagnostic assay capable of detecting and quantifying tumor antigens (e.g., MAGEB2) can help in the early detection and classification of cancer in patients, and can also predict the clinical response and outcome of appropriate cancer treatment.

[0004] The ability to reliably detect these tumor antigens may provide an early indicator of disease and / or disease progression. Immunodiagnostic assays are important tools for detecting various disease states, including cancer. However, such assays are not always sensitive and / or specific enough to reliably detect certain tumor antigens present on tumor cells, especially when their expression levels are low and / or not expressed on the surface of tumor cells.

[0005] In some instances, a molecular diagnostic assay is desired and may provide the required specificity and sensitivity and therefore be the best option for detecting a particular tumor antigen, however in other instances, it is desirable to confirm the appropriate expression of the tumor antigen within the tumor tissue sample and therefore an immunohistochemical assay may be more suitable. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Lee and Potts,J.Mol.Biol.,2018 Summary of the Invention [Problem to be solved by the invention]

[0007] Thus, there remains a need for sensitive and accurate diagnostic assays that detect tumor antigens useful for detecting malignant cells and / or that facilitate prediction of efficacy and improve safety of associated therapeutic approaches. [Means for solving the problem]

[0008] In one embodiment, the invention provides an isolated antigen-binding construct that binds to MAGEB2, wherein the isolated antigen-binding construct binds to an epitope comprising a sequence selected from SEQ ID NOs: 2, 3, 4 or 388-554.

[0009] In another embodiment, the invention provides an isolated antigen-binding construct comprising CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the sequence set forth in SEQ ID NO: 85, CDRL2 comprises the sequence set forth in SEQ ID NO: 86, CDRL3 comprises the sequence set forth in SEQ ID NO: 87, CDRH1 comprises the sequence set forth in SEQ ID NO: 229, CDRH2 comprises the sequence set forth in SEQ ID NO: 230, and CDRH3 comprises the sequence set forth in SEQ ID NO: 231.

[0010] In another embodiment, the invention provides an antigen-binding construct comprising CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the sequence set forth in SEQ ID NO: 73, CDRL2 comprises the sequence set forth in SEQ ID NO: 74, CDRL3 comprises the sequence set forth in SEQ ID NO: 75, CDRH1 comprises the sequence set forth in SEQ ID NO: 217, CDRH2 comprises the sequence set forth in SEQ ID NO: 218, and CDRH3 comprises the sequence set forth in SEQ ID NO: 219.

[0011] In another embodiment, the invention provides an isolated antigen-binding construct comprising CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the sequence set forth in SEQ ID NO: 91, CDRL2 comprises the sequence set forth in SEQ ID NO: 92, CDRL3 comprises the sequence set forth in SEQ ID NO: 93, CDRH1 comprises the sequence set forth in SEQ ID NO: 235, CDRH2 comprises the sequence set forth in SEQ ID NO: 236, and CDRH3 comprises the sequence set forth in SEQ ID NO: 237.

[0012] In a further embodiment, the present invention provides an isolated antigen-binding construct comprising a light chain variable region comprising the sequence set forth in SEQ ID NO: 346 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 347. In yet another embodiment, the present invention provides an isolated antigen-binding construct comprising a light chain variable region comprising the sequence set forth in SEQ ID NO: 338 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO: 339.

[0013] In another embodiment, the present invention provides an antigen-binding construct comprising a light chain variable region comprising the sequence set forth in SEQ ID NO:350 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:351.

[0014] In another embodiment, the present invention provides a method for producing an antibody that binds to MAGEB2, the method comprising immunizing an animal with a peptide comprising a sequence selected from SEQ ID NO: 2, 3 or 4, and isolating an antibody that binds to MAGEB2 from the animal.

[0015] In another embodiment, the invention provides a method of treating a tumor in a subject comprising obtaining from the subject a sample comprising cells derived from the tumor, determining that the subject will respond to treatment with an anti-MAGEB2 therapeutic agent by measuring the level of MAGEB2 in the sample using an antigen-binding construct provided herein and determining that the subject will respond to treatment with an anti-MAGEB2 therapeutic agent, and administering an effective amount of the anti-MAGEB2 therapeutic agent to the subject.

[0016] In another embodiment, the present invention provides a method of identifying a subject as being in need of an anti-MAGEB2 therapeutic comprising: a) determining the level of MAGEB2 in a sample obtained from the subject using an antigen-binding construct provided herein; and b) identifying the subject as being in need of an anti-MAGEB2 therapeutic if the level of MAGEB2 is increased compared to a control.

[0017] In another embodiment, the invention provides a method of determining a treatment for a subject having a MAGEB2 positive tumor comprising determining the level of MAGEB2 in a sample obtained from the subject using an antigen binding construct provided herein and determining that the treatment comprises an anti-MAGEB2 therapeutic if the level of MAGEB2 is increased compared to a control.In another embodiment, the invention provides a method of determining the effectiveness of treatment with an anti-MAGEB2 therapeutic in a subject comprising determining the level of MAGEB2 in a sample obtained from the subject before treatment with the anti-MAGEB2 therapeutic and after treatment with the anti-MAGEB2 therapeutic using an antigen binding construct provided herein and determining that the treatment is effective if the level of MAGEB2 positive tumor cells is reduced after treatment with the anti-MAGEB2 therapeutic.

[0018] In another embodiment, the present invention provides a method of diagnosing a subject having a tumor, comprising: a) determining the level of MAGEB2 in a sample obtained from the subject using an antigen-binding construct provided herein; and b) diagnosing the subject as having a MAGEB2 positive tumor if the level of MAGEB2 is increased compared to a control.

[0019] In another embodiment, the present invention provides a method for identifying a subject having a MAGEB2 positive tumor, the method comprising: a) determining the level of MAGEB2 in a sample obtained from the subject using an antigen-binding construct provided herein; and b) identifying the subject as having a MAGEB2 positive tumor if the level of MAGEB2 is increased compared to a control.

[0020] In another embodiment, the present invention provides a method of identifying a subject as being in need of an anti-MAGEB2 therapeutic comprising: a) determining the level of MAGEB2 in a sample obtained from the subject using an antigen-binding construct provided herein; and b) identifying the subject as being in need of an anti-MAGEB2 therapeutic if the level of MAGEB2 is increased compared to a control.

[0021] In another embodiment, the present invention provides a method of determining a treatment for a subject having a MAGEB2 positive tumor, comprising determining the level of MAGEB2 in a sample obtained from the subject using an antigen-binding construct provided herein, and determining that the treatment comprises an anti-MAGEB2 therapeutic agent if the level of MAGEB2 is increased compared to a control. [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 shows the gating hierarchy used to enrich for MAGEB2 N-terminal helix, N-terminal peptide and mid-loop region peptide binders as described in Example 2 herein. [Diagram 2]Shown are two plots showing binding by various anti-MAGEB2 antibodies binding to the immunogen peptide and full-length MAGEB2 protein. The MAGEB2 panel was run in a limited antigen style assay (only the highest concentration is shown) to identify the highest affinity binders to full-length MAGEB2 protein. [Diagram 3] Immunohistochemistry results of MAGEB2 IHC assay measuring immunoreactivity in MAGEB2 transfected cells. Strong MAGEB2 IHC staining was observed in CHO-MAGE-B2+ cells with four anti-MAGEB2 antibodies, 4G17, 1J15, 1C3, and 1I14, but not with the IgG control. [Figure 4] Immunohistochemistry results of MAGEB5 IHC assay measuring immunoreactivity in MAGEB2 transfected cells. No MAGEB2 IHC staining was observed in CHO-MAGEB5+ cells with the four anti-MAGEB2 antibodies 4G17, 1J15, 1C3, 1I14 and the IgG control. [Diagram 5] Immunohistochemistry results of MAGEB2 IHC assay measuring immunoreactivity in control testicular tissue are shown. Strong MAGEB2 IHC staining in spermatogonia was observed in testis with four anti-MAGEB2 antibodies 4G17, 1J15, 1C3, 1I14 at 2ug / ml, but not with the IgG control. Some nuclear staining was seen with antibodies 4G17, 1J15, 1C3, and Leydig cell staining was seen with antibody 1J15. [Figure 6] Immunohistochemistry results of MAGEB2 IHC assay measuring immunoreactivity in normal human tissues. Non-specific particulate staining in hepatocytes was seen with anti-MAGEB2 antibodies 4G17, 1J15, 1C3, but not with anti-MAGEB2 antibody 1I14 and IgG control in liver tissue cores (normal human tissue) at 5ug / ml. [Figure 7]Immunohistochemistry results of MAGEB2 IHC assay measuring immunoreactivity in testis and liver tissues with decreasing doses of 1C3 antibody. At 2ug / ml Ab concentration, the 1C3 clone of anti-MAGEB2 antibody has weak to moderate intensity specific staining in a significant proportion of spermatogonia, as previously observed. Nuclear staining is rare and the intensity is slightly weaker than that observed at higher concentrations. However, at this concentration, there is no nonspecific background staining in the liver. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] MAGEB2 is aberrantly expressed in various types of human cancer. MAGEB2 is not expressed on the surface of cells. However, MAGEB2 peptides are presented on the surface of tumor cells by MHC class I molecules. In particular, MAGEB2 peptide GVYDGEEHSV (SEQ ID NO: 1) is presented on the surface of tumor cells by MHC class I molecules as a peptide-MHC ("pMHC") complex. See, for example, US Patent Application Publication Nos. 2016 / 0250307A1 (US Patent Application No. 14 / 975,952) and 2017 / 0080070A1 (US Patent Application No. 15 / 357,757). MAGEB2 peptide-MHC complexes are presented on the surface of cells and are potentially targets for diagnostic agents, but the copy number of this peptide-MHC complex on the cell surface is too low to be detected. Thus, described herein are binding constructs (eg antibodies) that are capable of binding to and detecting MAGEB2 expressed in cells.

[0024] binding construct The present invention provides binding constructs comprising a domain that binds to a MAGEB2 protein. These binding constructs are also referred to as antigen-binding constructs.

[0025] The term binding construct comprises the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof, capable of binding to its specific target or antigen. Typically, a binding domain according to the invention comprises the minimum structural requirements of an antibody that allow binding of the target. This minimum requirement can for example be defined by the presence of at least three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region), preferably all six CDRs. An alternative approach to define the minimum structural requirements of an antibody is by definition of the epitope that the antibody binds to a protein domain of the target protein that constitutes, within the structure of the specific target, an epitope region (epitope cluster), respectively, or by reference to a specific antibody that competes with the epitope of the defined antibody. Alternatively, the minimum structural requirements can be defined by a paratope sequence within the binding domain of the antibody.

[0026] The binding construct of the present invention comprises at least one binding domain. The term "binding domain" in the context of the present invention characterizes a domain that specifically binds to, specifically interacts with or recognizes a given target epitope or a given target region on a target molecule, such as MAGEB2 or a specific region within MAGEB2. The structure and function of the binding domain is based on the structure and / or function of an antibody, such as the structure and / or function of a full-length or full immunoglobulin molecule, and is derived from the variable heavy (VH) and / or variable light (VL) domains of an antibody or a fragment thereof. In a particular embodiment, the binding domain comprises the presence of three light chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VL region) and / or three heavy chain CDRs (i.e. CDR1, CDR2 and CDR3 of the VH region). In a particular embodiment, the binding domain is generated or can be obtained by phage display or library screening methods, rather than by grafting CDR sequences from an existing (monoclonal) antibody onto a scaffold.

[0027] The binding domain of the binding construct according to the invention may, for example, comprise the CDRs mentioned above. Preferably, the CDRs are comprised within the framework of an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), but need not comprise both. An Fd fragment, for example, has two VH regions and often retains some antigen-binding function of the intact antigen-binding domain. Further examples of formats of antibody fragments, antibody variants or binding domains include (1) a Fab fragment, which is a monovalent fragment having the VL, VH, CL and CH1 domains; (2) a F(ab')2 fragment, which is a bivalent fragment having two Fab fragments linked by a disulfide bridge at the hinge region; (3) an Fd fragment having two VH and CH1 domains; (4) an Fv fragment having the VL and VH domains of a single arm of an antibody, (5) a dAb fragment having a VH domain (Ward et al., (1989) Nature 341:544-546); (6) isolated complementarity determining regions (CDRs), and (7) single chain Fvs (scFvs), the latter of which are preferred (e.g., from an scFV library). Examples of embodiments of binding constructs according to the invention are described, for example, in WO 00 / 006605, WO 2005 / 040220, WO 2008 / 119567, WO 2010 / 037838, WO 2013 / 026837, WO 2013 / 026833, US 2014 / 0308285, US 2014 / 0302037, WO 2014 / 144722, WO 2014 / 151910 and WO 2015 / 048272.

[0028] In certain embodiments of the invention, the binding construct is a full-length antibody as described herein below.

[0029] The definition of "binding domain" or "domain that binds" also includes fragments of full-length antibodies, such as VH, VHH, VL, (s)dAb, Fv, light chain (VL-CL), Fd (VH-CH1), heavy chain, Fab, Fab', F(ab')2 or "rIgG" (a "half antibody" consisting of a heavy and light chain). Binding constructs according to the invention may also include modified fragments of antibodies, also called antibody variants or antibody derivatives. Examples include scFv, di-scFv or bis(s)-scFv, scFv - Fc, scFv-zipper, scFab, Fab2, Fab3, diabody, single chain diabody, tandem diabody (Tandab), tandem di-scFv, tandem tri-scFv, "minibody" exemplified by structures such as: (VH-VL-CH3)2, (scFv - These include, but are not limited to, ((scFv)2-CH3+CH3), ((scFv)2-CH3) or (scFv-H3-scFv)2, multibodies, such as triabodies or tetrabodies, and single domain antibodies, such as nanobodies or single variable domain antibodies comprising only one variable region, which may be VHH, VH or VL, that specifically bind to an antigen or target independently of other variable regions or domains. Further possible formats of binding constructs according to the invention are crossbodies, maxibodies, hetero-Fc constructs, mono-Fc constructs and scFc constructs. Examples of these formats are described herein below.

[0030] According to the present invention, the binding domain is in the form of one or more polypeptides. Such polypeptides may contain proteinaceous and non-proteinaceous parts (e.g. chemical linkers or chemical cross-linking agents such as glutaraldehyde). Proteins (including fragments thereof, preferably biologically active fragments and peptides, usually having less than 30 amino acids) comprise two or more amino acids linked together via covalent peptide bonds (resulting in a chain of amino acids).

[0031] As used herein, the term "polypeptide" refers to a group of molecules that usually consists of more than 30 amino acids. Polypeptides may further form multimers, such as dimers, trimers and higher oligomers, i.e., may consist of multiple polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher order structures of such multimers are therefore referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is an antibody molecule, which in its naturally occurring form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide", "polypeptide" and "protein" also refer to naturally occurring modified peptides / polypeptides / proteins, which have been modified, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. As referred to herein, "peptide", "polypeptide" or "protein" may also be chemically modified, such as pegylation. Such modifications are well known in the art and are described herein below.

[0032] The definition of "antibody" according to the present invention includes full-length antibodies, including camelid antibodies and other immunoglobulins produced by biotechnological or protein engineering methods or processes. Such full-length antibodies may be, for example, monoclonal, recombinant, chimeric, deimmunized, humanized and human antibodies as well as antibodies from other species such as mouse, hamster, rabbit, rat, goat or non-human primate.

[0033] In the antibodies provided herein, the variable regions of the immunoglobulin chains generally exhibit the same overall structure, including relatively conserved framework regions (FR) linked by three hypervariable regions (more often called "complementarity determining regions" or CDRs). The CDRs from the two chains of each heavy / light chain pair described above are typically aligned by the framework regions to form a structure that specifically binds to the target epitope. From the N-terminus to the C-terminus, both naturally occurring light and heavy chain variable regions typically have these elements in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. A numbering system has been devised to assign numbers to the amino acids that occupy each position in these domains. This numbering system is defined in Kabat Sequences of Proteins of Immunological Interest (1987 and 1991, NIH, Bethesda, Md.) or Chothia & Lesk, 1987, J. Mol. Biol. 196:901-917; Chothia et al., 1989, Nature 342:878-883.

[0034] The term "variable" refers to that portion of an antibody or immunoglobulin domain (i.e., the "variable domain") that exhibits variability in sequence and is involved in determining the specificity and binding affinity of a particular antibody. The pairing of a variable heavy chain (VH) and a variable light chain (VL) together form the antigen-binding site.

[0035] The variability is not uniformly distributed throughout the variable domain of an antibody, but is concentrated in subdomains of each of the heavy and light chain variable regions. These subdomains are called "hypervariable regions" or "complementarity determining regions" (CDRs). The more conserved (i.e. non-hypervariable) parts of the variable domains are called "framework" regions (FRMs or FRs) and provide a scaffold for the six CDRs in the three-dimensional space that forms the antigen-binding surface. Naturally occurring heavy and light chain variable domains each contain four FRM regions (FR1, FR2, FR3 and FR4) that largely adopt a β-sheet configuration, connected by three hypervariable regions that form loop connections and sometimes form part of the β-sheet structure. The hypervariable regions of each chain are held together in close proximity by the framework regions and usually contribute to the formation of the antigen-binding site together with the hypervariable regions of the other chain.

[0036] The term "CDR" and its plural "CDRs" refer to the complementarity determining regions, three of which constitute the binding properties of the light chain variable region (CDR-L1, CDR-L2 and CDR-L3) and three of which constitute the binding properties of the heavy chain variable region (CDR-H1, CDR-H2 and CDR-H3). The CDRs contain most of the residues involved in the specific interaction of the antibody with the antigen and thus contribute to the functional activity of the antibody molecule, i.e., they are the main determinants of specific binding to a particular target.

[0037] The exact definition of the boundaries and lengths of CDRs are subject to various classifications and numbering systems. Thus, CDRs can be represented by Kabat, Chothia, contact, or any other boundary definition, including the numbering systems described herein. Although the boundaries are different, each of these systems has some overlap in the parts that constitute the so-called "hypervariable regions" in the variable sequences. Thus, the definition of CDRs according to these systems may differ in the length and the boundary regions with respect to the adjacent framework regions. See, e.g., Kabat (an approach based on interspecies sequence variability), Chothia (an approach based on crystallographic studies of antigen-antibody complexes) and / or MacCallum (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991; Chothia et al., J. MoI. Biol, 1987, 196:901-917; and MacCallum et al., J. Mol. Biol, 1996, 262:732). Yet another standard for characterizing antigen-binding sites is the AbM definition used by Oxford Molecular's AbM antibody modeling software. See, e.g., Protein Sequence and Structure Analysis of Antibody Variable Domains, Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). Two residue identification techniques can be combined to define hybrid CDRs, so long as they define overlapping, but non-identical, regions; however, the Kabat system of numbering is preferred.

[0038] Typically, CDRs form loop structures that can be classified as canonical structures. The term "canonical structure" refers to the main chain conformation that an antigen-binding (CDR) loop adopts. Each canonical structure can be characterized by the torsion angle of the polypeptide backbone. Corresponding loops between antibodies can therefore have very similar three-dimensional structures, despite the high amino acid sequence variability found in most of the loops (Chothia and Lesk, J. Mol. Biol., 1987, 196:901; Chothia et al., Nature, 1989, 342:877; Martin and Thornton, J. Mol. Biol, 1996, 263:800). Furthermore, there is a relationship between the adopted loop structure and the amino acid sequence surrounding it. The conformation of a particular canonical class is determined by the length of the loop and the amino acid residues present in key positions within the loop and within the conserved framework (i.e., outside the loop). Therefore, assignment to a particular canonical class can be made based on the presence of these key amino acid residues.

[0039] The term "canonical structure" may also include considerations regarding the linear sequence of an antibody, for example, as classified by Kabat (Kabat et al.). The Kabat numbering scheme is a widely adopted standard for numbering the amino acid residues of antibody variable domains in a consistent manner, and is the preferred scheme for application in the present invention, as mentioned elsewhere herein. Additional structural considerations may also be used to determine the canonical structure of an antibody. For example, differences not fully reflected by the Kabat numbering can be described by the Chothia et al. numbering system and / or revealed by other techniques, such as crystallography and two- or three-dimensional computer modeling. Thus, a given antibody sequence may be classified into a canonical class that allows, among other things, the identification of a suitable chassis sequence (e.g., based on the desire to include various canonical structures in a library). The Kabat numbering of antibody amino acid sequences and the structural considerations described in Chothia et al. (supra) and their significance for interpreting the canonical aspects of antibody structure are described in the literature. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known in the art. For a general overview of antibody structure, see Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, eds. Harlow et al., 1988.

[0040] As used herein, the term "single-chain Fv", "single-chain antibody" or "scFv" refers to a single polypeptide chain antibody fragment that contains both the heavy and light chain variable regions but lacks the constant region. Typically, single-chain antibodies further comprise a polypeptide linker between the VH and VL domains that enables them to form the desired structure that allows for antigen binding. Single-chain antibodies are discussed in detail by Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994). Various methods of making single chain antibodies are known, such as those described in U.S. Patent Nos. 4,694,778 and 5,260,203; International Patent Publication WO 88 / 01649; Bird (1988) Science 242:423-442; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; Ward et al. (1989) Nature 334:54454; Skerra et al. (1988) Science 242:1038-1041. In certain embodiments, single chain antibodies can be bispecific, multispecific, human and / or humanized, and / or synthetic.

[0041] In some embodiments, the binding construct of the present invention is an "in vitro generated binding construct". This term refers to a binding construct according to the above definition, in which all or part of the variable region (e.g. at least one CDR) is generated in the selection of non-immune cells, for example in vitro phage display, protein chips or any other method that allows testing of candidate sequences for their ability to bind to an antigen. In other embodiments, the binding construct sequence is generated by genomic rearrangement in immune cells of an animal. Thus, this term preferably excludes sequences that are generated exclusively by genomic rearrangement in immune cells of an animal. It is envisaged that the first and / or second domain of the binding construct can be generated or obtained by phage display or library screening methods, but not by grafting CDR sequences of existing (monoclonal) antibodies onto the scaffold.

[0042] A "recombinant antibody" is an antibody produced through the use of recombinant DNA technology or genetic engineering.

[0043] As used herein, the term "monoclonal antibody" (mAb) or monoclonal antibody construct refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., individual antibodies comprising the population that are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific and directed against a single antigenic site or epitope on the antigen, in contrast to conventional (polyclonal) antibody preparations that typically contain different antibodies directed against different antigenic sites or epitopes. In addition to their specificity, monoclonal antibodies are advantageous in that they are synthesized by a clonal cell culture and are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies and should not be construed as requiring production of the antibody by a particular method.

[0044] For the preparation of monoclonal antibodies, any technique that results in antibodies produced by continuous cell line cultures may be used. For example, the monoclonal antibodies used may be made by the hybridoma method first described by Koehler et al., Nature, 256:495 (1975) or by recombinant DNA techniques (see, for example, U.S. Pat. No. 4,816,567). Further examples of techniques for producing human monoclonal antibodies include the trioma technique, the human B-cell hybridoma technique (Kozbor, Immunology Today 4 (1983), 72) and the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc. (1985), 77-96).

[0045] The hybridomas can then be screened using standard methods such as enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance analysis, e.g., Biacore™, to identify one or more hybridomas that produce antibodies that specifically bind to a particular antigen. Any form of the relevant antigen can be used as the immunogen, for example, recombinant antigen, naturally occurring form, any variant or fragment thereof, and antigenic peptides thereof. Surface plasmon resonance, as employed in the Biacore system, can be used to increase the efficiency of binding of phage antibodies to epitopes of surface antigens of target cells (Schier, Human Antibodies Hybridomas 7 (1996), 97-105; Malmborg, J. Immunol. Methods 183 (1995), 7-13).

[0046] Another exemplary method for generating monoclonal antibodies includes screening protein expression libraries, such as phage display or ribosome display libraries, as described, for example, in Ladner et al., U.S. Pat. No. 5,223,409, Smith (1985) Science 228:1315-1317, Clackson et al., Nature, 352:624-628 (1991), and Marks et al., J. Mol. Biol., 222:581-597 (1991).

[0047] In addition to using display libraries, the relevant antigens can be used to immunize non-human animals, such as rodents (such as mice, hamsters, rabbits, or rats). In one embodiment, the non-human animals contain at least a portion of a human immunoglobulin gene. For example, mouse strains deficient in mouse antibody production can be engineered with large fragments of the human Ig (immunoglobulin) locus. Hybridoma technology can be used to produce and select antigen-specific monoclonal antibodies derived from genes with the desired specificity. See, for example, XENOMOUSE™, Green et al. (1994) Nature Genetics 7:13-21, US Patent Publication No. 2003-0070185, WO 96 / 34096, and WO 96 / 33735.

[0048] Monoclonal antibodies can also be obtained from non-human animals and subsequently modified, e.g., humanized, deimmunized, chimerized, etc., using recombinant DNA techniques known in the art. Examples of modified binding constructs include humanized variants of non-human antibodies, "affinity matured" antibodies (see, e.g., Hawkins et al. J. Mol. Biol. 254, 889-896 (1992) and Lowman et al., Biochemistry 30, 10832-10837 (1991)), and antibody variants with modified effector functions (see, e.g., U.S. Pat. No. 5,648,260; Kontermann and Duebel (2010), supra; and Little (2009), supra).

[0049] In immunology, affinity maturation is the process by which B cells produce antibodies with increased affinity to antigens during an immune response. Repeated exposure to the same antigen causes the host to produce antibodies with successively higher affinities. Similar to natural prototypes, in vitro affinity maturation is based on the principle of mutation and selection. In vitro affinity maturation has been successfully used to optimize binding constructs, such as antibody fragments. Random mutations within the CDRs are introduced using radiation, chemical mutagens or error-prone PCR. In addition, chain shuffling can increase genetic diversity. Two or three rounds of mutation and selection using display methods such as phage display usually result in antibody fragments with affinities in the low nanomolar range.

[0050] A preferred type of amino acid substitution mutation of the binding construct involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody). Generally, the resulting variants selected for further development will have improved biological properties relative to the parent antibody from which they were generated. A convenient method for generating such substitution variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants so generated are displayed in a monovalent form from filamentous phage particles as fusions with the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the binding domain and, for example, human MAGEB2. Such contact and adjacent residues are candidates for substitution by the techniques detailed herein. Having generated such variants, a panel of variants may be screened as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

[0051] The antibodies of the present invention include, inter alia, "chimeric" antibodies (immunoglobulins) in which a portion of, in particular, the heavy and / or light chains are identical or homologous to corresponding sequences in antibodies from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chains are identical or homologous to corresponding sequences in antibodies from another species or belonging to another antibody class or subclass, and fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that contain variable domain antigen-binding sequences derived from a non-human primate (e.g., Old World monkey, ape, etc.) and human constant region sequences. Various methods for producing chimeric antibodies have been described. See, e.g., Morrison et al., Proc. Natl. Acad. ScL USA 81:6851, 1985; Takeda et al., Nature 314:452, 1985; Cabilly et al., U.S. Pat. No. 4,816,567; Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., EP 0171496; EP 0173494; and GB 2177096.

[0052] A "humanized" antibody, variant or fragment thereof (Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequence of an antibody) is an antibody or immunoglobulin of mostly human sequence that contains minimal sequence derived from non-human immunoglobulin. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also referred to as CDR) of the recipient are replaced by residues from a hypervariable region of a non-human (e.g., rodent) species (donor antibody) such as mouse, rat, hamster or rabbit having the desired specificity, affinity, and capacity. In some cases, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, as used herein, "humanized antibodies" may also comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine and optimize antibody performance. A humanized antibody may also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature, 321:522-525 (1986); Reichmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992).

[0053] Humanized antibodies or fragments thereof can be generated by replacing sequences of Fv variable domains not directly involved in antigen binding with equivalent sequences from human Fv variable domains. Exemplary methods for generating humanized antibodies or fragments thereof are provided by Morrison (1985) Science 229:1202-1207; Oi et al. (1986) BioTechniques 4:214, and U.S. Pat. Nos. 5,585,089, 5,693,761, 5,693,762, 5,859,205, and 6,407,213. These methods include isolating, manipulating, and expressing nucleic acid sequences that encode all or part of immunoglobulin Fv variable domains from at least one of the heavy or light chains. Such nucleic acids can be obtained from hybridomas and other sources that produce antibodies against a predetermined target, as described above. The recombinant DNA encoding the humanized antibody molecule can then be cloned into an appropriate expression vector.

[0054] Humanized antibodies can also be made using transgenic animals, such as mice, that express human heavy and light chain genes but are unable to express endogenous mouse immunoglobulin heavy and light chain genes. Winter describes an exemplary CDR crafting method that can be used to prepare the humanized antibodies described herein (U.S. Pat. No. 5,225,539). All of the CDRs of a particular human antibody can be replaced with at least a portion of a non-human CDR, or only a portion of the CDRs can be replaced with a non-human CDR. It is only necessary to replace as many CDRs as are required for the binding of the humanized antibody to a given antigen.

[0055] Humanized antibodies can be optimized by introducing conservative substitutions, consensus sequence substitutions, germline substitutions and / or back mutations. Such modified immunoglobulin molecules can be generated by any of several techniques known in the art (e.g., Teng et al., Proc. Natl. Acad. Sci. USA, 80:7308-7312, 1983; Kozbor et al., Immunology Today, 4:7279, 1983; Olsson et al., Meth. Enzymol., 92:3-16, 1982, and EP 239400).

[0056] The terms "human antibody," "human antibody construct," and "human binding domain" include antibodies, antibody fragments, and binding domains having antibody regions, such as variable and constant regions or domains, that substantially correspond to human germline immunoglobulin sequences known in the art, including, for example, those described by Kabat et al. (1991) supra. The human antibodies, antibody fragments, or binding domains of the invention may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, particularly CDR3. Human antibodies, antibody fragments, or binding domains can have at least one, two, three, four, five, or more positions substituted with amino acid residues not encoded by human germline immunoglobulin sequences. The definitions of human antibodies, antibody fragments, and binding domains as used herein also contemplate fully human antibodies, which include only non-artificial and / or genetically modified human sequences of antibodies, which may be derived by using techniques or systems such as Xenomouse. Preferably, a "fully human antibody" does not include any amino acid residues that are not encoded by human germline immunoglobulin.

[0057] The ability to clone and reconstruct megabase-sized human loci in yeast artificial chromosomes YACs and introduce them into the mouse germline provides a powerful approach for elucidating the functional elements of very large or coarsely mapped loci and for generating useful models of human disease. Furthermore, the use of such techniques to replace mouse loci with their human equivalents will provide unique insight into the expression and regulation of nascent human gene products, their transfer to other systems, and their involvement in disease induction and progression.

[0058] An important practical application of such a strategy is the "humanization" of the mouse humoral immune system. The introduction of human immunoglobulin (Ig) loci into mice in which the endogenous Ig genes have been inactivated provides an opportunity to study the mechanisms underlying the programmed expression and assembly of antibodies and their role in B-cell development. Furthermore, such a strategy would provide an ideal source for the generation of fully human monoclonal antibodies (mAbs), which would represent a key milestone in realizing the potential of antibody therapy in human diseases. Fully human antibodies are expected to minimize the immunogenic and allergic reactions inherent to mouse or mouse-derived mAbs, thereby increasing the efficacy and safety of the administered antibodies / constructs. The use of fully human antibodies can be expected to provide substantial advantages in the treatment of chronic and recurrent human diseases such as inflammation, autoimmunity and cancer, which require repeated administration of compounds.

[0059] One approach towards this goal has been to engineer mouse strains deficient in mouse antibody production with large fragments of the human Ig loci, with the expectation that such mice would produce a broad repertoire of human antibodies without mouse antibodies. Large human Ig fragments would preserve the broad diversity of variable genes and the proper regulation of antibody production and expression. By utilizing the mouse machinery for antibody diversification and selection and the lack of immune tolerance to human proteins, the human antibody repertoires recapitulated in these mouse strains should produce high affinity antibodies against any antigen of interest, including human antigens. Using hybridoma technology, antigen-specific human mAbs with the desired specificity could be easily generated and selected. This general strategy was demonstrated in conjunction with the generation of the first XenoMouse mouse strains (see Green et al. Nature Genetics 7:13-21 (1994)). This XenoMouse strain was engineered with YACs containing 245 kb and 190 kb germline-configured fragments of the human heavy and kappa light chain loci, respectively, that contained the core sequences of the variable and constant regions. The human Ig-containing YACs proved compatible with the mouse system for both antibody rearrangement and expression, and were able to replace inactivated mouse Ig genes. This was demonstrated by their ability to induce B cell development to produce an adult-like human repertoire of fully human antibodies and to produce antigen-specific human mAbs. These results also suggested that the introduction of a large portion of the human Ig locus, containing multiple V genes, additional regulatory elements, and human Ig constant regions, could recapitulate a virtually complete repertoire characterized by the human humoral response to infection and immunization. Recently, extending the work of Green et al., the introduction of megabase-sized germline-configured YAC fragments of the human heavy and kappa light chain loci, respectively, introduced approximately 80% of the human antibody repertoire. See Mendez et al. Nature Genetics 15:146-156 (1997) and U.S. patent application Ser. No. 08 / 759,620.

[0060] The generation of XenoMouse® animals has been described in detail in U.S. patent application Ser. Nos. 07 / 466,008, 07 / 610,515, 07 / 919,297, 07 / 922,649, 08 / 031,801, 08 / 112,848, 08 / 234,145, 08 / 376,279, 08 / 430,938, 08 / 464,584, 08 / 464,582, 08 / 463,191, 08 / 462,837, 08 / 463,192, 08 / 462,837, 08 / 463,279, 08 / 463,303, 08 / 463,192, 08 / 463,279, 08 / 463,303, 08 / 463,303, 08 / 463,279 ...279, 08 / 463,303, 08 / 463,279, 08 / 463,279, 08 / 4 This is further discussed and detailed in U.S. Pat. Nos. 08 / 486,853, 08 / 486,857, 08 / 486,859, 08 / 462,513, 08 / 724,752 and 08 / 759,620, as well as U.S. Pat. Nos. 6,162,963, 6,150,584, 6,114,598, 6,075,181 and 5,939,598, and Japanese Patent Publications Nos. 3068180B2, 3068506B2 and 3068507B2. See also Mendez et al. Nature Genetics 15:146-156 (1997) and Green and Jakobovits J. Exp. Med. 188:483-495 (1998), EP 0463151 B1, WO 94 / 02602, WO 96 / 34096, WO 98 / 24893, WO 00 / 76310 and WO 03 / 47336.

[0061] In another approach, others including GenPharm International, Inc. have utilized a "minilocus" approach. In the minilocus approach, an exogenous Ig locus is mimicked by including fragments (individual genes) from the Ig locus. Thus, one or more VH genes, one or more DH genes, one or more JH genes, a mu constant region and a second constant region (preferably a gamma constant region) form a construct that is inserted into an animal. This approach is described in U.S. Pat. No. 5,545,807 to Surani et al., and U.S. Pat. Nos. 5,545,806, 5,625,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, 5,814,318, 5,877,397, 5,874,299, and 6,255,458, respectively, to Lonberg and Kay, U.S. Pat. Nos. 5,591,669 and 6,023.010 to Krimpenfort and Berns, and U.S. Pat. Nos. 5,591,669 and 6,023.010 to Berns et al. Nos. 5,612,205, 5,721,367, and 5,789,215 to Choi and Dunn, and U.S. Pat. No. 5,643,763 to GenPharm. and U.S. Patent Application Nos. 07 / 574,748, 07 / 575,962, 07 / 810,279, 07 / 853,408, 07 / 904,068, 07 / 990,860, 08 / 053,131, 08 / 096,762, 08 / 155,301, 08 / 161,739, 08 / 165,699, and 08 / 209,741 to International.See also EP 0546073B1, WO 92 / 03918, WO 92 / 22645, WO 92 / 22647, WO 92 / 22670, WO 93 / 12227, WO 94 / 00569, WO 94 / 25585, WO 96 / 14436, WO 97 / 13852 and WO 98 / 24884, and U.S. Pat. No. 5,981,175. See also Taylor et al. (1992), Chen et al. (1993), Tuaillon et al. (1993), Choi et al. (1993), Lonberg et al. (1994), Taylor et al. (1994), Tuaillon et al. (1995), and Fishwild et al. (1996).

[0062] Kirin has also demonstrated the production of human antibodies from mice into which large chromosome pieces or entire chromosomes have been introduced by microcell fusion. See EP 773288 and EP 843961. Xenerex Biosciences is developing a promising human antibody production technology in which SCID mice are reconstituted with human lymphoid cells, e.g., B and / or T cells. The mice can then be immunized with an antigen to generate an immune response against the antigen. See U.S. Pat. Nos. 5,476,996, 5,698,767 and 5,958,765.

[0063] In some embodiments, the binding construct of the present invention is an "isolated" or "substantially pure" binding construct. "Isolated" or "substantially pure" as used to describe the binding construct disclosed herein means a construct that has been identified, separated and / or recovered from components of its production environment. Preferably, the binding construct is free or substantially free from association with all other components from its production environment. Contaminating components of its production environment, such as components resulting from recombinant transfected cells, are materials that would normally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones and other proteinaceous or non-proteinaceous solutes. It is understood that an isolated protein may comprise a wide range of percent concentrations, for example, 5% to 99.9% by weight of the total protein content, depending on the circumstances. The polypeptide may be produced at significantly higher concentrations by using inducible or high expression promoters to produce high concentration levels. This definition includes the production of the binding construct in a variety of organisms and / or host cells known in the art. In a preferred embodiment, the binding construct is purified (1) sufficiently to obtain at least 15 residues of N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain. However, typically, an isolated binding construct will be prepared by at least one purification step.

[0064] Peptides are short chains of amino acid monomers linked by covalent peptide (amide) bonds. Thus, peptides are classified in the broad chemical class of biological oligomers and polymers. The amino acids that are part of a peptide or polypeptide chain may be referred to as "residues" and numbered consecutively. All peptides, except cyclic peptides, have an N-terminal residue at one end of the peptide and a C-terminal residue at the other end. Oligopeptides consist of very few amino acids (usually 2-20). Polypeptides are longer continuous unbranched peptide chains. Peptides are distinguished from proteins on the basis of size, and may be understood to include approximately 50 amino acids or less as an arbitrary criterion. Proteins usually consist of one or more polypeptides arranged in a biologically functional manner. Although aspects of the laboratory techniques applied to peptides versus polypeptides and proteins (e.g., details of electrophoresis, chromatography, etc.) differ, the size boundaries that distinguish peptides from polypeptides and proteins are not absolute. Thus, in the context of the present invention, the terms "peptide", "polypeptide" and "protein" may be used interchangeably, with the term "polypeptide" often being preferred.

[0065] Polypeptides may further form multimers (e.g., dimers, trimers and higher oligomers) consisting of multiple polypeptide molecules. The polypeptide molecules forming such dimers, trimers, etc. may be identical or non-identical. The corresponding higher order structures of such multimers are therefore referred to as homo- or heterodimers, homo- or heterotrimers, etc. An example of a heteromultimer is a full-length antibody or immunoglobulin molecule, which in its naturally occurring form consists of two identical polypeptide light chains and two identical polypeptide heavy chains. The terms "peptide", "polypeptide" and "protein" also refer to naturally occurring modified peptides / polypeptides / proteins, which have been modified, for example, by post-translational modifications such as glycosylation, acetylation, phosphorylation, etc. As referred to herein, a "peptide", "polypeptide" or "protein" may also be chemically modified, such as pegylation. Such modifications are well known in the art and are described herein below.

[0066] The binding construct binds to the antigen with a binding affinity of ≦10 as measured by surface plasma resonance technology (e.g., BIACore, GE-Healthcare Uppsala, Sweden) or kinetic exclusion assay (KinExA, Sapidyne, Boise, Idaho). -7 A binding construct is said to "specifically bind" or "immunospecifically bind" to an antigen if it binds with a dissociation constant (KD) of M. According to the present invention, a binding construct specifically binds or immunospecifically binds to MAGEB2.

[0067] However, due to sequence similarities between homologous proteins in different species, a binding construct or binding domain that specifically binds to its target (such as a human target) may cross-react with homologous target molecules from different species (e.g., from non-human primates). Thus, the term "specific / immunospecific binding" can include binding of a binding construct or binding domain to epitopes of more than one species or structurally related epitopes.

[0068] The term "epitope" refers to a region on an antigen or a specific amino acid residue to which a binding domain, such as an antibody or immunoglobulin or a derivative, fragment or variant of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic, and thus the term epitope is also sometimes referred to herein as an "antigenic structure" or "antigenic determinant". The binding domain is therefore the "antigen interaction site". Said binding / interaction is also understood to define "specific recognition".

[0069] An "epitope" can be formed by both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. A "linear epitope" is an epitope that comprises a contiguous primary amino acid sequence recognized as an epitope. A linear epitope generally comprises at least 3 or at least 4, more usually at least 5 or at least 6 or at least 7, and often from about 8 to about 10 amino acids or more than 10 amino acids in a unique sequence.

[0070] A "conformational epitope" is an epitope in which, in contrast to a linear epitope, the primary sequence of amino acids that comprise the epitope is not the only element that defines the recognized epitope (e.g., an epitope in which the primary sequence of amino acids is not necessarily recognized by the binding domain). Typically, a conformational epitope comprises a greater number of amino acids compared to a linear epitope and comprises a discontinuous amino acid sequence. With respect to the recognition of a conformational epitope, the binding domain paratope recognizes the three-dimensional structure of an antigen, preferably a peptide or protein or a fragment thereof (in the context of the present invention, the antigenic structure for one of the binding domains is contained within the target cell surface antigen protein). For example, when a protein molecule folds to form a three-dimensional structure, certain amino acids and / or polypeptide backbones that form a conformational epitope are juxtaposed, thereby allowing the antibody to recognize the epitope. Methods for determining the three-dimensional structure of an epitope include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, and site-specific spin labeling, and electron paramagnetic resonance (EPR) spectroscopy.

[0071] Various methods are known in the art for identifying proteins, regions of proteins or peptides that are useful as immunogens or in screening assays, in one embodiment, as described in the Examples herein, Shannon entropy analysis of the MAGEB2 protein amino acid sequence can identify specific peptide sequences that can be used as immunogens.

[0072] Exemplary MAGEB2 peptides identified for use as immunogens include MAGEB2 peptide aa43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide) (SEQ ID NO: 2), MAGEB2 peptide aa95-125: SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (MHD N-terminal helix peptide) (SEQ ID NO: 3), MAGEB2 peptide aa185-200: DLTDEESLLSSWDFPR (MHD mid-loop peptide) (SEQ ID NO: 4).

[0073] Thus, in one embodiment of the present invention there is provided a method of immunising an animal comprising administering to the animal any of the peptides of SEQ ID NO: 2, 3 or 4.

[0074] In another embodiment, a method of producing an isolated antibody is provided, comprising immunizing an animal with any of the peptides of SEQ ID NO:2, 3 or 4 and isolating said antibody. In a further embodiment, a method of producing an isolated monoclonal antibody is provided, comprising immunizing an animal with any of the peptides of SEQ ID NO:2, 3 or 4 and further producing the monoclonal antibody using art-recognized steps as described herein.

[0075] In yet another embodiment, an isolated antibody produced by a process comprising immunizing an animal with any of the peptides of SEQ ID NO:2, 3 or 4 is provided.

[0076] In yet other embodiments, a fragment of the peptide of SEQ ID NO: 2, 3 or 4 may be used as an immunogen. Thus, in one embodiment of the invention, the peptide immunogen comprises a fragment of MAGEB2 peptide aa43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide) (SEQ ID NO: 2) consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids.

[0077] In another embodiment of the invention, the peptide immunogen comprises a fragment of MAGEB2 peptide aa95-125:SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (MHD N-terminal helical peptide) (SEQ ID NO: 3) consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0078] Another embodiment of the invention comprises a fragment of MAGEB2 peptide aa185-200:DLTDEESLLSSWDFPR (MHD mid-loop peptide) (SEQ ID NO: 4) consisting of 10, 11, 12, 13, 14 or 15 amino acids.

[0079] In a further embodiment of the invention, there is provided a binding construct which competes for binding with any of the binding constructs described in the invention. Competitive assays are well known in the art and exemplary assays are further described herein.

[0080] In other embodiments, alternative peptides that can be used as immunogens for screening are provided below in Table 1. Thus, the present invention provides Table 1.

[0081] [Table 1]

[0082] [Table 2]

[0083] [Table 3]

[0084] [Table 4]

[0085] [Table 5]

[0086] affinity The interaction between a binding domain and an epitope or an epitope-containing region means that the binding domain exhibits a measurable affinity for the epitope and / or epitope-containing region on a particular protein or antigen, and, unless otherwise specified, does not exhibit significant binding or reactivity to proteins or antigens other than MAGEB2. This affinity can be measured by a variety of techniques known to those skilled in the art, such as surface plasmon resonance assays such as Biacore assays or cell-based assays.

[0087] "Measurable affinity" is about 10 -6 M(KD) or greater. Preferably, the binding affinity is about 10 -12 ~10 -8 M, 10 -12 ~10 -9 M, 10 -12 ~10 -10 M, 10 -11 ~10 -8 M, preferably about 10 -11 ~10 -9 Binding is considered specific if it is M. Whether a binding domain specifically reacts with or binds to a target can easily be tested, inter alia, by comparing the reaction of said binding domain with a target protein or antigen with the reaction of said binding domain with proteins or antigens other than MAGEB2. Preferably, the binding domains of the invention do not essentially or substantially bind to proteins or antigens other than MAGEB2.

[0088] specificity The term "does not bind significantly" means that the binding construct or binding domain of the invention does not bind to proteins or antigens other than MAGEB2. For example, a binding construct or binding domain that exhibits binding to proteins with similar amino acid sequences, such as MAGEA4 or MAGEA8, is not a desirable binding construct.

[0089] In the present invention, the MAGEB2 binding constructs have a surprising level of specificity and selectivity for their target as evidenced by the lack of binding to target negative cells, see, e.g., Examples 2 and 3 herein.

[0090] This specificity and selectivity are highly desirable, yet difficult to obtain, properties of binding constructs in diagnostic assays, as they limit, reduce or eliminate off-target binding and any potential misreading.

[0091] Specific binding is believed to be mediated by specific motifs within the amino acid sequences of the binding domain and the antigen. Thus, binding occurs as a result of their primary, secondary and / or tertiary structure and as a result of secondary modifications of said structure. Specific interaction of the antigen interaction site with its specific antigen may result in simple binding of said site to the antigen. Furthermore, specific interaction of the antigen interaction site with its specific antigen may alternatively or additionally result in the initiation of a signal, for example by inducing a conformational change in the antigen, oligomerization of the antigen, etc.

[0092] MAGEB2-binding constructs The present invention provides binding constructs comprising a domain that binds to MAGEB2, in particular embodiments to aa43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide) (SEQ ID NO: 2), aa95-125: SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (MHD N-terminal helix peptide) (SEQ ID NO: 3) or aa185-200: DLTDEESLLSSWDFPR (SEQ ID NO: 4) of the MAGEB2 peptide.

[0093] Table 2 below provides exemplary MAGEB2 binding constructs, amino acid sequences of the VH-CDRs and VL-CDRs. Table 3 below provides exemplary MAGEB2 binding constructs, amino acid sequences of the VH domains and VL domains.

[0094] [Table 6]

[0095] [Table 7]

[0096] In one embodiment the invention provides an isolated binding domain that binds to MAGEB2, the binding domain comprising: a) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 229, CDR-H2 as set forth in SEQ ID NO: 230, and CDR-H3 as set forth in SEQ ID NO: 231, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 85, CDR-L2 as set forth in SEQ ID NO: 86, and CDR-L3 as set forth in SEQ ID NO: 87; or b) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 217, CDR-H2 as set forth in SEQ ID NO: 218 and CDR-H3 as set forth in SEQ ID NO: 219, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 73, CDR-L2 as set forth in SEQ ID NO: 74 and CDR-L3 as set forth in SEQ ID NO: 75; or c) A VH region comprising CDR-H1 as set forth in SEQ ID NO: 235, CDR-H2 as set forth in SEQ ID NO: 236, and CDR-H3 as set forth in SEQ ID NO: 237, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93.

[0097] In one embodiment the invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, the binding domain comprising: a) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 229, CDR-H2 as set forth in SEQ ID NO: 230, and CDR-H3 as set forth in SEQ ID NO: 231, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 85, CDR-L2 as set forth in SEQ ID NO: 86, and CDR-L3 as set forth in SEQ ID NO: 87; or b) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 217, CDR-H2 as set forth in SEQ ID NO: 218 and CDR-H3 as set forth in SEQ ID NO: 219, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 73, CDR-L2 as set forth in SEQ ID NO: 74 and CDR-L3 as set forth in SEQ ID NO: 75; or c) A VH region comprising CDR-H1 as set forth in SEQ ID NO: 235, CDR-H2 as set forth in SEQ ID NO: 236, and CDR-H3 as set forth in SEQ ID NO: 237, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93.

[0098] In one embodiment the invention provides an isolated binding domain that binds to MAGEB2, which binding domain binds to the same epitope as an antibody or binding construct comprising: a) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 229, CDR-H2 as set forth in SEQ ID NO: 230, and CDR-H3 as set forth in SEQ ID NO: 231, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 85, CDR-L2 as set forth in SEQ ID NO: 86, and CDR-L3 as set forth in SEQ ID NO: 87; or b) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 217, CDR-H2 as set forth in SEQ ID NO: 218 and CDR-H3 as set forth in SEQ ID NO: 219, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 73, CDR-L2 as set forth in SEQ ID NO: 74 and CDR-L3 as set forth in SEQ ID NO: 75; or c) A VH region comprising CDR-H1 as set forth in SEQ ID NO: 235, CDR-H2 as set forth in SEQ ID NO: 236, and CDR-H3 as set forth in SEQ ID NO: 237, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93.

[0099] In one embodiment the invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, which binding domain binds to the same epitope as an antibody or binding construct comprising: a) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 229, CDR-H2 as set forth in SEQ ID NO: 230, and CDR-H3 as set forth in SEQ ID NO: 231, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 85, CDR-L2 as set forth in SEQ ID NO: 86, and CDR-L3 as set forth in SEQ ID NO: 87; or b) a VH region comprising CDR-H1 as set forth in SEQ ID NO: 217, CDR-H2 as set forth in SEQ ID NO: 218 and CDR-H3 as set forth in SEQ ID NO: 219, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 73, CDR-L2 as set forth in SEQ ID NO: 74 and CDR-L3 as set forth in SEQ ID NO: 75; or c) A VH region comprising CDR-H1 as set forth in SEQ ID NO: 235, CDR-H2 as set forth in SEQ ID NO: 236, and CDR-H3 as set forth in SEQ ID NO: 237, and a VL region comprising CDR-L1 as set forth in SEQ ID NO: 91, CDR-L2 as set forth in SEQ ID NO: 92, and CDR-L3 as set forth in SEQ ID NO: 93.

[0100] In one embodiment the invention provides an isolated binding domain that binds to MAGEB2, the binding domain comprising: a) a VH region comprising SEQ ID NO: 347 and a VL region comprising SEQ ID NO: 346; or b) a VH region comprising SEQ ID NO: 339 and a VL region comprising SEQ ID NO: 338; or c) a VH region comprising SEQ ID NO: 351 and a VL region comprising SEQ ID NO: 350.

[0101] In one embodiment the invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, the binding domain comprising: a) a VH region comprising SEQ ID NO: 347 and a VL region comprising SEQ ID NO: 346; or b) a VH region comprising SEQ ID NO: 339 and a VL region comprising SEQ ID NO: 338; or c) a VH region comprising SEQ ID NO: 351 and a VL region comprising SEQ ID NO: 350.

[0102] In one embodiment the invention provides an isolated binding domain that binds to MAGEB2, which binding domain binds to the same epitope as an antibody or binding construct comprising: a) a VH region comprising SEQ ID NO: 347 and a VL region comprising SEQ ID NO: 346; or b) a VH region comprising SEQ ID NO: 339 and a VL region comprising SEQ ID NO: 338; or c) a VH region comprising SEQ ID NO: 351 and a VL region comprising SEQ ID NO: 350.

[0103] In one embodiment the invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, which binding domain binds to the same epitope as an antibody or binding construct comprising: a) a VH region comprising SEQ ID NO: 347 and a VL region comprising SEQ ID NO: 346; or b) a VH region comprising SEQ ID NO: 339 and a VL region comprising SEQ ID NO: 338; or c) a VH region comprising SEQ ID NO: 351 and a VL region comprising SEQ ID NO: 350.

[0104] In another embodiment, the present invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, wherein the binding domain comprises a VH region selected from the group consisting of any VH region and the corresponding VL region as set forth in any of the sequences in Tables 21-23 herein.

[0105] In another embodiment, the present invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, which binding domain binds to the same epitope as an antibody or binding construct comprising a VH region selected from the group consisting of any VH region and the corresponding VL region as set forth in any of the sequences in Table 23 herein.

[0106] In another embodiment, the present invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, the binding domain comprising CDR-H1, CDR-H2, CDR-H3 of a VH region selected from the group consisting of any VH region as set forth in any of the sequences in Table 22 herein, and further comprising the corresponding CDR-L1, CDR-L2, CDR-L3 of a VL region selected from the group consisting of any VL region as set forth in any of the sequences in Table 21 herein.

[0107] In another embodiment, the present invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, which binding domain comprises CDR-H1, CDR-H2, CDR-H3 of a VH region selected from the group consisting of any VH region as set out in any of the sequences in Table 22 herein, and further binds to the same epitope as an antibody or binding construct comprising the corresponding CDR-L1, CDR-L2, CDR-L3 of a VL region selected from the group consisting of any VL region as set out in any of the sequences in Table 21 herein.

[0108] In another embodiment, the present invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, wherein the binding domain comprises a VH and VL region consensus sequence selected from any of the sequences in Table 25 herein.

[0109] In another embodiment, the present invention provides an isolated binding construct comprising a binding domain that binds to MAGEB2, the binding domain comprising CDR-H1, CDR-H2, CDR-H3 of a VH region consensus sequence selected from the group consisting of any VH region as set forth in any of the sequences in Table 25 herein, and further comprising the corresponding CDR-L1, CDR-L2, CDR-L3 of a VL region consensus sequence selected from the group consisting of any VL region as set forth in any of the sequences in Table 25 herein.

[0110] The full sequence of an exemplary binding construct is shown below. 1C3_LC (sequence number 555) [ka] 1C3_HC (SEQ ID NO: 556) [ka] 1I14_LC (SEQ ID NO: 557) [ka] 1I14_HC (SEQ ID NO: 558) [ka] 1H17_LC (SEQ ID NO:559) [ka] 1H17_HC (SEQ ID NO:560) [ka]

[0111] competitive binding Whether an antibody or binding construct competes with another given antibody or binding construct for binding to an antigen (such as MAGEB2) can be measured in a competitive assay, such as a competitive ELISA. Avidin-conjugated microparticles (beads) can also be used. Each of these beads, when reacted with biotinylated proteins, can be used as a substrate on which the assay can be performed, similar to an avidin-coated ELISA plate. The antigen is coated on the beads, which are then pre-coated with the first antibody. A secondary antibody is added and any further binding is determined. The readout is performed by flow cytometry. Preferably, a cell-based competitive assay is used, using cells that naturally express MAGEB2 or cells stably or transiently transformed with MAGEB2, allowing binding to MAGEB2 expressed in cells. The term "compete for binding" in this context means that there is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80% or at least 90% competition between two test antibodies as determined by any one of the assays disclosed above, preferably a cell-based assay.

[0112] Competitive antibody binding assays include assays that determine the competitive binding of two antibodies / binding constructs to a cell surface-bound antigen. A common method is to detect the binding of two antibodies / binding constructs A and B to the same antigen expressed by a cell: blocking antigen by pre-incubating cells with antibody / binding construct A, followed by submaximal addition of labeled antibody / binding construct B and detecting binding of B compared to binding in the absence of A; titrating (i.e. adding different amounts) antibody / binding construct A in the presence of a submaximal amount of labeled antibody / binding construct B and detecting the effect on binding of B; or co-titrating A and B, incubating both antibodies / binding constructs together at maximal concentrations and detecting whether the total binding is equal to or exceeds the binding of A or B alone. i.e. a method that may not be affected by the order or relative amounts of addition of antibodies / binding constructs.

[0113] When two antibodies / binding constructs A and B compete for antigen, the antibodies will very often compete with each other in blocking assays regardless of the order of antibody addition. In other words, competition will be detected if the assay is performed in either direction. However, this is not always the case, and in some circumstances, the order of antibody addition or the direction of the assay may affect the signal generated. This may be due to differences in affinity or avidity of potentially competing antibodies / binding constructs. If the order of addition significantly affects the signal generated, it is concluded that the two antibodies / binding constructs compete if competition is detected in at least one order.

[0114] Epitope amino acid residues Structural analysis of the interaction between the target polypeptide and, for example, the binding domain of the construct contemplated herein provides the amino acid residues of the target epitope involved in the binding interaction. In certain embodiments, a crystal structure of the target-binder interaction can provide these amino acid residues. In other embodiments, various other analyses can be performed to identify the amino acid residues involved in the binding interaction, such as Xscan, alanine scanning, arginine scanning, and other techniques known to those skilled in the art. In the present invention, the binding constructs are made using specific peptide immunogens and therefore must bind to these specific peptide sequences.

[0115] In embodiments where a peptide is used as an immunogen, such as the peptides provided herein in SEQ ID NO: 2, 3 or 4, the isolated antibody is expected to bind to an epitope that includes all of the peptide immunogen or a fragment thereof. The epitope may be a linear epitope that includes all of the peptide immunogen amino acid sequence or a fragment thereof. Alternatively, and more likely for longer peptide immunogens, the epitope may be a conformational epitope that includes discontinuous amino acid sequences from within the peptide immunogen. Thus, in one embodiment of the present invention, the epitope to which the binding construct (e.g., an antibody) binds includes the MAGEB2 peptide aa43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide) (SEQ ID NO: 2).

[0116] In another embodiment, the epitope to which the binding construct (e.g., an antibody) binds comprises a fragment of MAGEB2 peptide aa43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide) (SEQ ID NO: 2) consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or 33 amino acids.

[0117] In another embodiment, the epitope to which the binding construct (e.g. an antibody) binds comprises at least two, at least three, at least four or at least five non-contiguous amino acid sequence fragments of the MAGEB2 peptide aa43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide) (SEQ ID NO: 2), each non-contiguous amino acid sequence fragment consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0118] In another embodiment of the invention, the epitope to which the binding construct of the invention binds comprises the MAGEB2 peptide aa95-125: SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (MHD N-terminal helical peptide) (SEQ ID NO: 3).

[0119] In another embodiment, the epitope to which the binding construct (e.g., an antibody) binds comprises a fragment of MAGEB2 peptide aa95-125:SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (MHD N-terminal helical peptide) (SEQ ID NO: 3) consisting of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids.

[0120] In another embodiment, the epitope to which the binding construct (e.g., an antibody) binds comprises at least two, at least three, at least four or at least five non-contiguous amino acid sequence fragments of the MAGEB2 peptide aa95-125:SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (MHD N-terminal helical peptide) (SEQ ID NO: 3), each non-contiguous amino acid sequence fragment consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[0121] In another embodiment of the invention, the epitope to which the binding construct of the invention binds comprises the MAGEB2 peptide aa185-200: DLTDEESLLSSWDFPR (MHD mid-loop peptide) (SEQ ID NO: 4).

[0122] In another embodiment, the epitope to which the binding construct (eg, an antibody) binds comprises a fragment of MAGEB2 peptide aa185-200:DLTDEESLLSSWDFPR (MHD mid-loop peptide) (SEQ ID NO: 4) consisting of 10, 11, 12, 13, 14 or 15 amino acids.

[0123] In another embodiment, the epitope to which the binding construct (e.g. an antibody) binds comprises at least two, at least three, at least four or at least five non-contiguous amino acid sequence fragments of the MAGEB2 peptide aa185-200:DLTDEESLLSSWDFPR (MHD mid-loop peptide) (SEQ ID NO: 4), each non-contiguous amino acid sequence fragment consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids.

[0124] In one embodiment, the invention provides a binding construct that binds to an epitope comprising any of the peptides of SEQ ID NOs: 2, 3, 4 or 388-554.

[0125] The region of the binding domain that binds to the epitope is called the "paratope". Specific binding is thought to be mediated by specific motifs within the amino acid sequences of the binding domain and the antigen. Thus, binding occurs as a result of their primary, secondary and / or tertiary structure and potential secondary modifications of said structure.

[0126] The term "Fc portion" or "Fc monomer" in the context of the present invention means a polypeptide comprising at least one domain having the function of a CH2 domain and at least one domain having the function of a CH3 domain of an immunoglobulin molecule. As is evident from the term "Fc monomer", a polypeptide comprising those CH domains is a "polypeptide monomer". An Fc monomer comprises a fragment of the constant region of an immunoglobulin excluding at least the first constant region immunoglobulin domain (CH1) of the heavy chain, but retaining at least a functional part of one CH2 domain and a functional part of one CH3 domain, which may be a polypeptide amino-terminal to the CH3 domain.

[0127] In one embodiment of this definition, an Fc monomer can be a polypeptide constant region comprising an Ig-Fc hinge region, a CH2 region and a portion of a CH3 region, the hinge region being amino-terminal to the CH2 domain. It is envisioned that the hinge region of the present invention promotes dimerization. Such an Fc polypeptide molecule can be obtained, for example, but not limited to, by papain digestion of an immunoglobulin region (which of course results in a dimer of two Fc polypeptides). In another aspect of this definition, an Fc monomer can be a polypeptide region comprising a CH2 region and a portion of a CH3 region. Such an Fc polypeptide molecule can be obtained, for example, but not limited to, by pepsin digestion of an immunoglobulin molecule.

[0128] In one embodiment, the polypeptide sequence of the Fc monomer is substantially similar to the Fc polypeptide sequence of the IgG1 Fc region, IgG2 Fc region, IgG3 Fc region, IgG4 Fc region, IgM Fc region, IgA Fc region, IgD Fc region, and IgE Fc region. (See, e.g., Padlan, Molecular Immunology, 31(3), 169-217 (1993)). Because some variants exist between immunoglobulins, and simply for clarity, the Fc monomer refers to the last two heavy chain constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three heavy chain constant region immunoglobulin domains of IgE and IgM. As stated, the Fc monomer may also include a flexible hinge N-terminal to these domains. In the case of IgA and IgM, the Fc monomer may include a J chain. In the case of IgG, the Fc portion comprises the immunoglobulin domains CH2 and CH3 and the hinge between the first two domains and CH2.

[0129] Although the boundaries of the Fc moiety may vary, an example of a human IgG heavy chain Fc moiety comprising a functional hinge, the CH2 and CH3 domains can be defined as comprising, for example, residues D231 to P476 (of the hinge domain - corresponding to D234 in Table 4 below), and L476 (for IgG4) at the carboxyl terminus of the CH3 domain, respectively (wherein the numbering is according to Kabat). Two Fc moieties or Fc monomers fused to each other via a peptide linker define a third domain of the binding construct of the invention, which may also be defined as an scFc domain.

[0130] In one embodiment of the present invention, it is envisaged that the scFc domains disclosed herein, the Fc monomers respectively fused to each other, are comprised only in the third domain of the binding construct. In some embodiments, the IgG hinge region can be identified by analogy using the Kabat numbering as shown in Table 4. In line with the above, for the hinge domains / regions of the present invention, the minimum requirement is envisaged to comprise amino acid residues corresponding to the IgG1 sequence stretch of D231 D234 to P243 according to the Kabat numbering. Similarly, it is envisaged that the hinge domains / regions of the present invention comprise or consist of the IgG1 hinge sequence DKTHTCPPCP (SEQ ID NO: 4) (corresponding to the stretch D234 to P243 as shown in Table 4 below - variations of said sequence are also envisaged if the hinge region still promotes dimerization). In a preferred embodiment of the present invention, the glycosylation site at Kabat position 314 of the CH2 domain in the third domain of the binding construct is removed by a N314X substitution (where X is any amino acid other than Q). The substitution is preferably a N314G substitution. In a more preferred embodiment, the CH2 domain comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0131] [Table 8]

[0132] In a further embodiment of the invention, the hinge domain / region comprises or consists of the IgG2 subtype hinge sequence ERKCCVECPPCP (SEQ ID NO: 5), the IgG3 subtype hinge sequence ELKTPLDTTHTCPRCP (SEQ ID NO: 6) or ELKTPLGDTTHTCPRCP (SEQ ID NO: 7) and / or the IgG4 subtype hinge sequence ESKYGPPCPSCP (SEQ ID NO: 8). The IgG1 subtype hinge sequence may be one of the following EPKSCDKTHTCPPCP (SEQ ID NO: 9). These core hinge regions are therefore also envisaged in the context of the present invention.

[0133] The locations and sequences of the IgG CH2 and IgG CH3 domains can be identified by similarity using the Kabat numbering set forth in Table 5.

[0134] [Table 9]

[0135] In one embodiment of the invention, the amino acid residues highlighted in bold within the CH3 domain of the first Fc monomer or both Fc monomers are deleted.

[0136] In classical full-length antibodies or immunoglobulins, each light (L) chain is linked to a heavy (H) chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. The heavy chain constant (CH) domain closest to the VH is usually called CH1. The constant ("C") domains are not directly involved in antigen binding, but exhibit various effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) and complement activation (complement-dependent cytotoxicity, CDC). The Fc region of an antibody is the "tail" region of a classical antibody that interacts with cell surface receptors called Fc receptors and several proteins of the complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the second and third constant domains (CH2 and CH3) of the two heavy chains of the antibody. The Fc region of IgM and IgE contains three heavy chain constant domains (CH2, CH3 and CH4) in each polypeptide chain. The Fc region also contains part of the so-called "hinge" region held together by one or more disulfides and non-covalent interactions. The Fc region of naturally occurring IgG carries highly conserved N-glycosylation sites. Glycosylation of the Fc fragment is essential for Fc receptor-mediated activity.

[0137] In some embodiments of the invention, the CH2 domain of one or preferably each (both) polypeptide monomer of the third domain comprises an intradomain cysteine ​​disulfide bridge. As known in the art, the term "cysteine ​​disulfide bridge" refers to a functional group having the general structure RSSR. This bond, also called SS bond or disulfide bridge or cysteine ​​clamp, is induced by the binding of two thiol groups of cysteine ​​residues. In certain embodiments, the cysteines that form the cysteine ​​disulfide bridge in the mature antibody construct are introduced into the amino acid sequence of the CH2 domain corresponding to 309 and 321 (Kabat numbering). In other embodiments, the cysteine ​​clamp is introduced in other domains of the binding construct. See also, for example, US Patent Application Publication No. 2016 / 0193295.

[0138] In one embodiment of the invention, the glycosylation site at Kabat position 314 of the CH2 domain is removed. This removal of glycosylation site is preferably achieved by an N314X substitution, where X is any amino acid except Q. Said substitution is preferably N314G. In a more preferred embodiment, said CH2 domain comprises the following substitutions (positions according to Kabat): V321C and R309C (these substitutions introduce intradomain cysteine ​​disulfide bridges at Kabat positions 309 and 321).

[0139] Covalent modifications of the binding construct are also included within the scope of the invention and are generally, but not always, performed post-translationally. For example, some types of covalent modifications of the binding construct are introduced into the molecule by reacting specific amino acid residues of the binding construct with organic derivatizing agents capable of reacting with selected side chains or N- or C-terminal residues.

[0140] Cysteinyl residues most commonly are reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives.

[0141] Cysteinyl residues are also derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole.

[0142] Histidyl residues are derivatized by reaction with diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide is also useful, and the reaction is preferably carried out in 0.1 M sodium cacodylate at pH 6.0. Lysinyl and amino terminal residues react with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of lysinyl residues. Other suitable reagents for derivatizing alpha-amino-containing residues include imidoesters such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactions with glyoxylate.

[0143] Arginyl residues are modified by reaction with one or more conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Due to the high pKa of the guanidine functional group, derivatization of arginine residues requires that the reaction be carried out under alkaline conditions. Moreover, these reagents can react with lysine groups and the arginine epsilon-amino group.

[0144] The specific modification of tyrosyl residues may be undertaken for the purpose of introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidazole and tetranitromethane are used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. To prepare labeled proteins for use in radioimmunoassay, tyrosyl residues are iodinated using 125I or 131I, the chloramine T method described above being suitable.

[0145] Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N=C=N-R'), where R and R' are optionally different alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0146] Derivatization with bifunctional agents is useful for crosslinking the binding constructs of the present invention to water-insoluble support matrices or support surfaces for use in various methods. Commonly used crosslinking agents include homobifunctional imidoesters including 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters such as esters with 4-azidosalicylic acid, disuccinimidyl esters such as 3,3-dithiobis(succinimidyl propionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate produce photoactivatable intermediates that can form crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and reactive substrates described in U.S. Pat. Nos. 3,969,287, 3,691,016, 4,195,128, 4,247,642, 4,229,537, and 4,330,440 are utilized for protein immobilization. Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues, respectively. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues is within the scope of the present invention.

[0147] Other modifications include hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine and histidine side chains (TECreighton, Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, pp. 79-86), acetylation of the N-terminal amine and amidation of any C-terminal carboxyl group.

[0148] Another type of covalent modification of binding constructs included within the scope of the present invention involves altering the glycosylation pattern of a protein. As is known in the art, glycosylation patterns can depend on both the sequence of the protein (e.g., the presence or absence of specific glycosylated amino acid residues, discussed below) or the host cell or organism in which the protein is produced. Particular expression systems are discussed below.

[0149] Glycosylation of polypeptides is usually either N-linked or O-linked. N-linked refers to the attachment of a sugar chain to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of a sugar chain to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0150] Addition of glycosylation sites to the binding construct is conveniently accomplished by altering the amino acid sequence to include one or more of the above tripeptide sequences (for N-linked glycosylation sites). Modifications may also be made by addition to or substitution of one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For ease of use, the amino acid sequence of the binding construct is preferably altered by changes at the DNA level, in particular by mutating the DNA encoding the polypeptide at preselected bases so that codons that translate into the desired amino acids are generated.

[0151] Another means of increasing the number of carbohydrate moieties on a conjugation construct is by chemical or enzymatic coupling of glycosides to proteins. These procedures are advantageous in that they do not require production of the protein in a host cell that has glycosylation capabilities for N- and O-linked glycosylation. Depending on the mode of linkage used, sugars can be attached to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as in cysteine, (d) free hydroxyl groups, such as in serine, threonine, or hydroxyproline, (e) aromatic residues, such as in phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330 and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0152] Removal of carbohydrate moieties present on the starting conjugation construct can be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment cleaves most or all sugars except the attached sugar (N-acetylglucosamine or N-acetylgalactosamine) while leaving the polypeptide intact. Chemical deglycosylation is described by Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exoglycosidases as described by Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites can be prevented by the use of the compound tunicamycin, as described by Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin blocks the formation of protein-N-glycosidic bonds.

[0153] Other modifications of the binding construct are also contemplated herein. For example, another type of covalent modification of the binding construct includes linking the binding construct to various non-proteinaceous polymers, including but not limited to various polyols such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol, in the manner described in U.S. Pat. Nos. 4,640,835, 4,496,689, 4,301,144, 4,670,417, 4,791,192, or 4,179,337. Furthermore, amino acid substitutions can be made at various positions within the binding construct to facilitate the addition of polymers such as, for example, PEG, as is known in the art.

[0154] In some embodiments, the covalent modification of the binding constructs of the present invention includes the addition of one or more labels. To reduce potential steric hindrance, the labeling group may be attached to the binding construct via a spacer arm of various lengths. A variety of methods for labeling proteins are known in the art and can be used in practicing the present invention. The term "label" or "labeling group" refers to any detectable label. In general, labels are divided into various classes depending on the assay in which they will be detected, examples of which include, but are not limited to: radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 I), magnetic labels (e.g., magnetic particles), redox-active moieties, optical dyes (including, but not limited to, chromophores, fluorophores and fluorophores), such as fluorescent groups (e.g., FITC, rhodamine, lanthanide fluorophores), chemiluminescent groups and fluorophores, which can be "small molecule" fluorophores or proteinaceous fluorophores, enzymes (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), biotinylation groups, predetermined polypeptide epitopes recognized by a secondary reporter (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).

[0155] "Fluorescent label" refers to any molecule that can be detected by its inherent fluorescent properties. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarins, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade BlueJ, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon Green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-Phycoerythrin (PE) (Molecular Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland.

[0156] Suitable proteinaceous fluorescent labels include, but are not limited to, GFPs of Renilla, Ptilosarcus or Aequorea species (Chalfie et al., 1994, Science 263:802-805), green fluorescent proteins including EGFP (Clontech Laboratories, Inc., Genbank Accession No. U55762), blue fluorescent proteins (BFP, Quantum Biotechnologies, Inc. 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H 1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), enhanced yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), luciferase (Ichiki et al., 1993, J. Immunol. 150:5408-5417), β-galactosidase (Nolan et al. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607) and Renilla (WO 92 / 15673, WO 95 / 07463, WO 98 / 14605, WO 98 / 26277, WO 99 / 49019, U.S. Pat. Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, and 5,925,558).

[0157] Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally discovered in a variety of different proteins and have since been identified in several DNA-binding proteins (Landschulz et al., 1988, Science 240:1759). Among the known leucine zippers are naturally occurring peptides and their derivatives that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94 / 10308, and a leucine zipper derived from pulmonary surfactant protein D (SPD) is described in Hoppe et al., 1994, FEBS Letters 344:191. The use of modified leucine zippers that allow stable trimerization of heterologous proteins fused thereto is described in Fanslow et al., 1994, Semin. Immunol. 6:267-78.

[0158] The binding constructs of the present invention may also comprise additional domains, for example, useful for the isolation of the molecule or for the adaptation of the pharmacokinetic profile of the molecule. The domains useful for the isolation of the binding construct may be selected from peptide motifs or secondarily introduced moieties that can be captured by an isolation method, for example, an isolation column. Non-limiting examples of such additional domains include peptide motifs known as Myc-tag, HAT-tag, HA-tag, TAP-tag, GST-tag, chitin binding domain (CBD-tag), maltose binding protein (MBP-tag), Flag-tag, Strep-tag and variants thereof (e.g., StrepII-tag) and His-tag. All of the binding constructs disclosed herein may comprise a His-tag domain, commonly known as a repeat of consecutive His residues, preferably five, more preferably six His residues (hexahistidine) in the amino acid sequence of the molecule. The His-tag may, for example, be located at the N-terminus or C-terminus of the binding construct, preferably at the C-terminus. Most preferably, a hexa-histidine tag (HHHHHH) (SEQ ID NO: 30) is linked to the C-terminus of the conjugate construct according to the present invention via a peptide bond. In addition, the PLGA-PEG-PLGA conjugate system can be combined with a polyhistidine tag for sustained release applications and improved pharmacokinetic profiles.

[0159] Diagnostic methods The binding constructs provided herein are useful for detecting MAGEB2 in biological samples and can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or pathologies associated with MAGEB2.

[0160] The disclosed binding constructs provide a means to detect the presence of MAGEB2 in a sample, for example, using classical immunohistological methods known to those of skill in the art (e.g., Tijssen, 1993, Practice and Theory of Enzyme Immunoassays, Vol 15 (Eds RH Burdon and PH van Knippenberg, Elsevier, Amsterdam); Zola, 1987, Monoclonal Antibodies: A Manual of Techniques, pp. 147-158 (CRC Press, Inc.); Jalkanen et al., 1985, J. Cell. Biol. 101:976-985; Jalkanen et al., 1987, J. Cell Biol. 105:3087-3096). Detection of MAGEB2 can be performed in vivo or in vitro, but is preferably performed in vitro on a sample comprising cells obtained from a patient. The method used to detect MAGEB2 expression in human tissue, such as tumor tissue, can be qualitative, semi-quantitative or quantitative. For example, art-recognized antibody-based methods for detecting protein levels in biological samples include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, electrochemiluminescence (ECL) assay, surface plasmon resonance, Western blot, immunoprecipitation, fluorescence-activated cell sorting (FACS), immunofluorescence, immunohistochemistry, and the like. The method of detecting MAGEB2 expression in biological samples can include control samples (negative and positive controls). For example, the negative control sample can be a sample that does not contain MAGEB2 protein, and the positive control sample is a sample that contains MAGEB2 protein. The presence or absence of MAGEB2 in a biological sample can be confirmed by comparing the results with the negative and positive controls.

[0161] In certain embodiments, the binding constructs can be used in immunohistochemistry (IHC) assays. These IHC assays can be performed on modern, high-throughput platforms, some of which are automated, such as the Roche-Ventana BenchMark ULTRA system.

[0162] IHC is a commonly used assay that involves the use of a binding construct, such as an antibody, that specifically binds to a target antigen in a tissue sample, thereby allowing the assay user to determine whether the target antigen is present in the tissue sample.

[0163] Immunohistochemical methods are well known in the art and are described, for example, in Immunohistochemical Staining Methods 6th Edition 2013, Taylor CR, Rudbeck L, eds., Dako North America (available as an educational guide "Immunohistochemical Staining Method guide" at www.dako.com). Immunohistochemical staining can be performed on fixed or frozen tissue sections. When using fixed tissue sections, such as formalin-fixed paraffin-embedded (FFPE) tissue sections, the procedure can generally use the following exemplary steps: obtaining a tumor tissue sample (e.g., by biopsy), fixing the tumor sample, embedding (e.g., in paraffin); sectioning and mounting; antigen retrieval; incubation with a primary antibody (e.g., a MAGEB2 antibody as described herein), detection (e.g., after amplification of the antigen / antibody complex signal) and interpretation by a skilled professional (e.g., using an art-recognized scoring system).

[0164] Suitable non-limiting fixatives include, for example, paraformaldehyde, glutaraldehyde, formaldehyde, acetic acid, acetone, osmium tetroxide, chromate, mercuric chloride, picric acid, alcohol (e.g., methanol, ethanol), Gender's solution, Rossman's solution, B5 fixative, Bouin's solution, Carnoy's fixative, and methacin. In one embodiment, the tumor sample is fixed in formaldehyde (e.g., 10% neutral buffered formalin, which corresponds to 4% formaldehyde in the buffer solution). Once fixed, the tumor sample may be dehydrated sequentially using alcohol or xylene, embedded in paraffin, and cut with a microtome to generate tumor tissue sections (e.g., having a thickness of about 4-5 μm), which may then be mounted on a microscope slide (e.g., adhesive-coated glass slide). Exemplary embedding materials include paraffin, celloidin, OCT, and methacin. TM Compounds, agar, plastic, or acrylic. Another exemplary material is Histogel (ThermoFisher Scientific), which contains agarose / glycerin, as described in the Examples herein.

[0165] Provided herein is a method of detecting MAGEB2 expression in a biological sample, such as a human tumor tissue sample, by contacting the sample with an anti-MAGEB2 antigen-binding construct, such as an anti-MAGEB2 antibody comprising the heavy and light chain variable region CDR sequences described herein or the heavy and light chain variable region sequences described herein, which antigen-binding construct specifically binds to human MAGEB2, and detecting binding of the antibody to MAGEB2 in the sample, for example by immunohistochemistry.

[0166] Thus, the present invention provides a method for diagnosing a subject having a tumor, the method comprising: a) determining the level of MAGEB2 in a sample obtained from the subject; and b) diagnosing the subject as having a MAGEB2-positive tumor if the level of MAGEB2 is increased compared to a control.

[0167] Provided herein is a method for identifying a subject having a MAGEB2-positive tumor, the method comprising: a) determining the level of MAGEB2 in a sample obtained from the subject; and b) identifying the subject as having a MAGEB2-positive tumor if the level of MAGEB2 is increased compared to a control.

[0168] Provided herein is a method for identifying a subject in need of an anti-MAGEB2 therapeutic agent, the method comprising: a) determining the level of MAGEB2 in a sample obtained from the subject; and b) identifying the subject as having a MAGEB2-positive tumor if the level of MAGEB2 is increased compared to a control.

[0169] Provided herein is a method for determining a treatment for a subject having a MAGEB2-positive tumor, the method comprising: a) determining the level of MAGEB2 in a sample obtained from the subject; and b) determining that the treatment includes an anti-MAGEB2 therapeutic agent if the level of MAGEB2 is increased compared to a control.

[0170] In certain embodiments, exemplary biological samples include blood samples, serum samples, cells, surgically removed tissues, and biopsy tissues (e.g., cancer tissues) obtained from cancer patients. Biological samples for use in the methods described herein can be fresh, frozen, or fixed samples. Biological samples, such as tumor samples, can be obtained from patients using routine methods, including, but not limited to, biopsy, surgical resection, or aspiration.

[0171] The expression of MAGEB2 protein in tissue, e.g., tumor samples, can be detected using direct or indirect methods. For example, in assays in which the binding construct is an antibody, the primary antibody may comprise a detectable moiety, such as the enzyme horseradish peroxidase (HRP) or a fluorescent label (e.g., FITC, TRITC), as described herein. In certain embodiments, the primary antibody does not itself comprise a detectable moiety, but instead is detected by a secondary antibody binding to it for indirect immunohistochemistry. Thus, in certain embodiments, the secondary antibody comprises a detectable label, e.g., an enzymatic, chromogenic or fluorescent label. In certain embodiments, the primary antibody is a chimeric antibody comprising a human variable region sequence and a non-human Fc region. A secondary antibody can be used to recognize the non-human Fc region of the primary antibody to reduce background staining.

[0172] In other embodiments, the avidin-biotin complex method (ABC method) can be used to detect MAGEB2 protein expression. In these embodiments, for example, when the binding construct is an antibody, the secondary antibody can be biotinylated and serve as a bridge between the primary antibody and the biotin-avidin-peroxidase complex. Other suitable non-limiting methods for immunohistochemistry include those described in Chapter 6 of the Immunohistochemical Staining Method guide, such as those described in Chilosi et al., Biotech Histochem 1994;69:235; Sabattini et al., J Clin Pathol 1998;51:506-11; and Gross et al., JBC 1959;234:1622.

[0173] General methods for preparing and staining frozen tissue sections are well known in the art and are described, for example, in Immunohistochemical Staining Method guide, Chapter 3 of Immunohistochemistry and Methods (Buchwalow and Bocker, Springer-Verlag Berlin Heidelberg 2010) and Chapter 21 of Theory and Practice of Histological Techniques (Bancroft and Gamble, 6.sup.th Edition, Elsevier Ltd., 2008). In some embodiments, slides stained with, for example, an anti-MAGEB2 antibody as described herein, can be further counterstained, for example with hematoxylin and / or eosin, using methods well known in the art.

[0174] MAGEB2 protein expression detected by immunohistochemistry can be digitally scanned and analyzed (e.g., as described in the Examples herein) or can be otherwise assessed and scored using art-recognized scoring methods, non-limiting examples of which are described below.

[0175] For example, in certain embodiments, MAGEB2 expression can be assessed using the H-score (histochemical score) system, which is widely used in the art and is useful given its dynamic range and use of weighted percentiles. The H-score is a semi-quantitative scoring system based on the following formula: 3× percentage of strongly stained cells (3+ staining)+2× percentage of moderately stained (2+ staining) cells+1× percentage of weakly stained (1+ staining) cells+0× percentage of unstained (0 staining) cells (score range is 0-300). See, e.g., McCarty et al., Cancer Res 1986;46:4244-8; Bosman et al., J Clin Pathol 1992;45:120-4; Dieset et al., Analyt Quant Cytol Histol 1996;18:351-4.

[0176] Control tissues may include, for example, matched non-tumor tissues from the same subject. Thus, in certain embodiments, the number of cells that stain positively for MAGEB2 using the binding constructs (e.g., antibodies) herein and the intensity of staining may be used to determine the H-score.

[0177] In other embodiments, MAGEB2 expression can be assessed using the Allred scoring system (Allred et al., Mod Pathol 1998;11:155-68; Harvey et al., J Clin Oncol 1999;17:1474-91). This scoring system involves adding a percentage score and an intensity score to obtain a total score. Thus, in some embodiments, a percentage score is obtained based on the estimated percentage of tumor cells that are positive for MAGEB2 (0: none, 1: <1 / 100, 2: 1 / 100 to 1 / 10, 3: 1 / 10 to 1 / 3, 4: 1 / 3 to 2 / 3, and 5: >2 / 3), and an intensity score is obtained based on the average intensity of MAGEB2 expression in positive tumor cells (0: none, 1: weak, 2: intermediate, 3: strong). The Allred score ranges from 0 to 8, with scores in the range of 3 to 8 being considered positive (i.e., positive detection). Thus, in certain embodiments, tumors with an all-red score of 3 to 8 for MAGEB2 staining, such as 3, 4, 5, 6, 7 or 8, are considered MAGEB2 positive tumors.

[0178] In other embodiments, the scoring system may be automated, e.g., computerized, and quantified by image analysis, as described herein above. Automation methods are well known in the art. For example, an average threshold measurement (ATM) score, which obtains an average of 255 staining intensity levels, can be calculated, e.g., as described in Choudhury et al., J Histochem Cytochem 2010;58:95-107, Rizzardi et al., Diagnostic Pathology 2012;7:42-52. Another automated scoring system is AQUA® (Automated Quantitative Analysis), which is performed, e.g., using tissue microarrays (TMA) on a continuous scale. AQUA® is a hybrid of standard immunohistochemistry and flow cytometry that provides an objective numerical score ranging from 1 to 255, and includes antigen retrieval, the use of primary and secondary antibodies, and multiplexed fluorescent detection. Optimal cut-off points can be determined as described in Camp et al. Clin Cancer Res 2004;10:7252-9. The AQUA® scoring system is described in detail in Camp et al., Nat Med 2002;8:1323-7; Camp et al., Cancer Res 2003;63:1445-8; Ghosh et al., Hum Pathol 2008;39:1835-43; Bose et al., BMC Cancer 2012;12:332; Mascaux et al., Clin Cancer Res 2011;17:7796-807. Other suitable automated immunohistochemistry platforms include commercially available platforms such as the Leica BOND RX staining platform. The platform detects target protein expression in tumor tissue based on staining intensity on the following scale: minimal, <1 cell per 20x objective field; mild, 1-10 cells per 20x objective field; moderate, 10-50 cells per 20x objective field; severe, 50-200 cells per 20x objective field; and severe, >200 cells per 20x objective field.

[0179] In some embodiments, a tumor is considered MAGEB2 positive and is likely to benefit from a therapeutic agent that targets MAGEB2 if a sample of the tumor has more MAGEB2 positive cells than a matched normal tissue from the same cancer patient.

[0180] In certain embodiments, if the number of MAGEB2 positive cells in a tumor sample exceeds a threshold level, the cancer patient is likely or predicted to benefit from a therapeutic agent targeting MAGEB2. The threshold level may be a minimum level of MAGEB2 as determined by a given detection system, for example immunohistochemistry as further described herein, and in certain embodiments, if the number of MAGEB2 positive cells in a tumor sample exceeds a threshold number or percentage, using the art-recognized scoring system described above, the cancer patient is likely or predicted to benefit from a therapeutic agent targeting MAGEB2. Thus, in some embodiments, a sample can be defined as "MAGEB2 positive" if the number or percentage of MAGEB2 positive cells exceeds a certain threshold number or percentage, using the scoring system described herein.

[0181] In certain embodiments, a tumor sample in which at least 1%, for example at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90% or at least 95% of cells within the tumor express MAGEB2 is said to be "MAGEB2 positive" and indicates that a cancer patient is likely or predicted to respond to a therapeutic agent targeted to MAGEB2.

[0182] In some embodiments, an H-score can be calculated for a tumor sample. Thus, in certain embodiments, a tumor sample having an H-score of at least 5, such as at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, at least 250, at least 275, or at least 290, is considered "MAGEB2 positive" and indicates that a cancer patient is likely or predicted to respond to a therapeutic agent that targets MAGEB2.

[0183] In some embodiments, an All-Red score can be calculated for the tumor sample. Thus, in certain embodiments, a tumor sample having an All-Red score of at least 3, such as at least 4, at least 5, at least 6, at least 7, or 8, is considered "MAGEB2 positive" and indicates that the cancer patient is likely or predicted to respond to a therapeutic agent that targets MAGEB2.

[0184] In some embodiments, an AQUA score can be calculated. Thus, in certain embodiments, a tumor sample having an AQUA score of at least 5, such as at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 125, at least 150, at least 175, at least 200, at least 225, or at least 250, is considered "MAGEB2 positive" and indicates that the cancer patient is likely or predicted to respond to a therapeutic agent that targets MAGEB2.

[0185] In certain embodiments, a combination of one of the above scoring systems and another detection modality, such as FISH, is used to increase the accuracy of determining whether a tumor is MAGEB2 positive or MAGEB2 negative.

[0186] In certain embodiments, the patient is diagnosed with squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, squamous non-small cell lung cancer (NSCLC), non-squamous NSCLC, glioma, gastrointestinal cancer, renal cancer (e.g., clear cell carcinoma), ovarian cancer, liver cancer, colorectal cancer, endometrial cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), prostate cancer (e.g., hormone refractory prostate adenocarcinoma), thyroid cancer, neuroblastoma, pancreatic cancer, glioblastoma (glioblastoma multiforme), cervical cancer, stomach cancer, bladder cancer, liver cancer, breast cancer, colon cancer, head and neck cancer (or carcinoma), gastric cancer, cancer), germ cell tumors, childhood sarcomas, sinonasal natural killer, melanoma (e.g., metastatic melanoma, such as cutaneous or intraocular malignant melanoma), bone cancer, skin cancer, uterine cancer, anal cancer, testicular cancer, fallopian tube cancer, endometrial cancer, cervical cancer, vaginal cancer, vulvar cancer, esophageal cancer, small intestine cancer, endocrine system cancer, parathyroid cancer, adrenal gland cancer, soft tissue sarcoma, urethral cancer, penile cancer, childhood solid tumors, ureteral cancer, renal pelvis cancer, central nervous system (CNS) cancer Neoplasms, primary CNS lymphomas, tumor angiogenesis, spinal axis tumors, brain tumors, brain stem gliomas, pituitary adenomas, Kaposi's sarcoma, epidermoid carcinoma, squamous cell carcinoma, T-cell lymphomas, environmentally induced cancers including asbestos-induced cancers, virus-associated cancers or cancers of viral origin (e.g., human papillomavirus (HPV-associated or HPV-origin tumors)), hematological malignancies derived from the two major blood cell lineages, i.e., myeloid cell lineage (producing granulocytes, erythrocytes, platelets, macrophages and mast cells) or lymphoid cell lineage (producing B cells, T cells, NK cells and plasma cells), e.g., all types of leukemias, lymphomas and myelomas, e.g., acute, chronic, lymphocytic and / or myeloid leukemias (e.g., acute leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CL ... leukemia (CLL), chronic myeloid leukemia (CML), anaplastic AML (M0), myeloblastic leukemia (M1), myeloblastic leukemia (M2; with cellular maturation), promyelocytic leukemia (M3 or M3 variant [M3V]), myelomonocytic leukemia (M4 or M4 variant [M4E] with eosinophilia), monocytic leukemia (M5), erythroleukemia (M6), megakaryoblastic leukemia (M7), isolated granulocytic sarcoma and chloroma;Lymphomas (e.g., Hodgkin's lymphoma (HL), non-Hodgkin's lymphoma (NHL), B-cell hematological malignancies, e.g., B-cell lymphoma, T-cell lymphoma, lymphoplasmacytoid lymphoma, monocytoid B-cell lymphoma, mucosa-associated lymphoid tissue (MALT) lymphoma, anaplastic (e.g., Ki 1+) large cell lymphoma, adult T-cell lymphoma / leukemia, mantle cell lymphoma, angioimmunoblastic T-cell lymphoma, hemocentric lymphoma, intestinal T-cell lymphoma, primary mediastinal B-cell lymphoma, precursor T-lymphoblastic lymphoma, T-lymphoblastic lymphoma / leukemia (T-Lbly / T-ALL), peripheral T-cell lymphoma, lymphoblastic lymphoma, post-transplant lymphoproliferative disorder, true histiocytic lymphoma, primary central nervous system lymphoma , primary effusion lymphoma, B-cell lymphoma, lymphoblastic lymphoma (LBL), lymphoid hematopoietic neoplasms, acute lymphoblastic leukemia, diffuse large B-cell lymphoma, Burkitt's lymphoma, follicular lymphoma, diffuse histiocytic lymphoma (DHL), immunoblastic large cell lymphoma, precursor B-lymphoblastic lymphoma, cutaneous T-cell lymphoma (CTLC) (also called mycosis fungoides or Sézary syndrome) and Waldenstrom's lymphoma. lymphoplasmacytoid lymphoma (LPL) with macroglobulinemia; myeloma (e.g., IgG myeloma, light chain myeloma, nonsecretory myeloma, smoldering myeloma (also called indolent myeloma), solitary plasmacytoma, multiple myeloma, chronic lymphocytic leukemia (CLL), hairy cell lymphoma); hematopoietic tumors of myeloid lineage, tumors of mesenchymal origin including fibrosarcoma and rhabdomyosarcoma; central nervous system tumors including seminoma, teratocarcinoma, astrocytoma, and schwannoma tumors of the nervous and peripheral nerves; tumors of mesenchymal origin including fibrosarcoma, rhabdomyosarcoma and osteosarcoma; other tumors including melanoma, xeroderma pigmentosum, keratoderma, seminoma, thyroid follicular carcinoma and teratocarcinoma; hematopoietic tumors of the lymphatic system, such as T-cell and B-cell tumors (e.g., T-cell disorders such as T-prolymphocytic leukemia (T-PLL), including but not limited to small cell and encephalo-like cell types); large granular lymphocytic leukemia (LGL), preferably of the T-cell type; a / d T-NHL hepatosplenic lymphoma; peripheral / post-thymic T-cell lymphoma (pleomorphic and immunoblastic subtypes);The patient has a cancer selected from the group consisting of angiocentric (nasal) T-cell lymphoma, head and neck cancer, renal cancer, rectal cancer, thyroid cancer, acute myeloid lymphoma, and any combination of the foregoing. In certain embodiments, the cancer is metastatic, refractory, or recurrent cancer;

[0187] Patient treatment Once a cancer patient is identified as likely to benefit from treatment with a therapeutic agent targeted to MAGEB2-expressing tumors (i.e., having a MAGEB2-positive tumor), the patient can be treated with an anti-MAGEB2 therapeutic agent (e.g., an anti-MAGEB2 immunotherapeutic agent, such as those found in U.S. patent application Ser. No. 63 / 027,148, entitled "MAGEB2 Binding Constructs," filed May 19, 2020, and U.S. patent application Ser. No. 63 / 128,773, entitled "MAGEB2 Binding Constructs," filed December 21, 2020). Thus, provided herein is a method of treating a tumor in a subject, comprising determining that the subject will respond to treatment with an anti-MAGEB2 therapeutic agent by obtaining a sample from the subject comprising cells derived from the tumor, measuring the level of MAGEB2 in the sample, and determining that the subject will respond to treatment with an anti-MAGEB2 therapeutic agent, and administering an effective amount of an anti-MAGEB2 therapeutic agent to the subject.

[0188] Provided herein is a method of treating a cancer patient, the method comprising: (a) determining whether a tumor of a cancer patient is MAGEB2 positive using a method described herein, for example by contacting a tumor sample from the patient with a binding construct described herein, where the antibody or antigen-binding fragment thereof specifically binds to human MAGEB2, detecting binding of the antibody to MAGEB2 in the sample, and determining the level of MAGEB2 protein expression in the sample, where a level of MAGEB2 protein in tumor cells that exceeds a threshold level indicates that the tumor is MAGEB2 positive; and (b) if the tumor is determined to be MAGEB2 positive, administering a therapeutically effective amount of an agent that targets and kills cells expressing MAGEB2.

[0189] Provided herein is a method of treating a tumor in a subject, wherein the subject's tumor has been tested for MAGEB2 levels, and the subject's tumor is MAGEB2 positive, the method comprising administering an anti-MAGEB2 therapeutic agent to the subject.

[0190] Provided herein is a method for determining the effectiveness of treatment with an anti-MAGEB2 therapeutic agent in a subject, the method comprising determining the level of MAGEB2 in a sample obtained from the subject before treatment with the anti-MAGEB2 therapeutic agent and after treatment with the anti-MAGEB2 therapeutic agent, and determining that the treatment is effective if the level of MAGEB2 positive tumor cells decreases after treatment with the anti-MAGEB2 therapeutic agent.

[0191] Provided herein is a method of monitoring treatment with an anti-MAGEB2 therapeutic agent in a subject, the method comprising determining the level of MAGEB2 in a sample obtained from the subject at a first time point, determining the level of MAGEB2 in a sample obtained from the subject at a second time point, and optionally continuing treatment with the anti-MAGEB2 therapeutic agent if the level of MAGEB2 at the second time point is decreased compared to the level at the first time point.

[0192] Provided herein is a method for identifying a subject as responding to treatment with an anti-MAGEB2 therapeutic agent, the method comprising determining the level of MAGEB2 in a sample obtained from the subject, and identifying the subject as responding to treatment with the anti-MAGEB2 therapeutic agent if the level of MAGEB2 is reduced upon treatment with the anti-MAGEB2 therapeutic agent.

[0193] In specific embodiments of the above embodiments, the subject or patient is treated with a therapeutically effective amount of any of the molecules disclosed in U.S. Patent Application No. 63 / 027,148, filed May 19, 2020, and entitled "MAGEB2 Binding Constructs," and U.S. Patent Application No. 63 / 128,773, filed December 21, 2020, the contents of which are incorporated herein by reference.

[0194] In further specific embodiments of the above embodiment, various reference levels of MAGEB2 in patient samples can be compared to each other. For example, a first reference level obtained at a first time point can be compared to a second level obtained at a second time point. Furthermore, for example, MAGEB2 levels can be compared before and after drug treatment. In this example, it can be assumed that successful treatment with a drug will result in a decrease in the MAGEB2 level measured after treatment compared to the reference MAGEB2 level measured before treatment. The first and second tissue samples can be obtained, for example, within 3-7 days, 1-3 weeks, or 1-3 months of each other or even longer.

[0195] In certain embodiments, a method for monitoring MAGEB2-expressing tumors in cancer patients comprises: (a) detecting MAGEB2 protein expression in a tissue (e.g., tumor) sample at a first time point by using an anti-MAGEB2 antibody or an antigen-binding portion thereof; (b) determining the level of MAGEB2 protein expression at the first time point; (c) detecting MAGEB2 protein expression in a tissue (e.g., tumor, which may be the same tumor as biopsied at the first time point) sample at a second time point using the same antibody used in step (a); and (d) determining the level of MAGEB2 protein expression in the tumor at the second time point.

[0196] A method for monitoring the effectiveness of anti-MAGEB2 immunotherapy (e.g., one of the molecules provided in U.S. Patent Application No. 63 / 027,148, filed May 19, 2020, and U.S. Patent Application No. 63 / 128,773, filed December 21, 2020, and entitled "MAGEB2 Binding Constructs") in a patient having a MAGEB2-positive tumor, comprising: (a) detecting MAGEB2 protein expression in the MAGEB2-positive tumor at a first time point before or after initiating anti-MAGEB2 immunotherapy by using an anti-MAGEB2 antibody; (b) determining the level of MAGEB2 protein expression in the tumor at the first time point; and (c) detecting MAGEB2 protein expression in the MAGEB2-positive tumor at a second time point after initiating anti-MAGEB2 immunotherapy using the same antibody as in step (a). (d) determining the level of MAGEB2 protein expression in the tumor at a second time point; (e) comparing the levels of MAGEB2 protein expression determined at the first and second time points, wherein a higher level at the first time point compared to the second time point may indicate that anti-MAGEB2 immunotherapy is effective, a lower score at the first time point compared to the second time point may indicate that anti-MAGEB2 immunotherapy is ineffective, and an unchanged score at the first time point compared to the second time point may indicate that anti-MAGEB2 immunotherapy is stabilised. MAGEB2 expression may be determined in a tumor, tumor tissue or tumor cells.

[0197] In certain embodiments, the status of a MAGEB2-expressing tumor may be monitored repeatedly after initial diagnosis (i.e., after the tumor is determined to be MAGEB2 positive), e.g., 1 month after initial diagnosis, 2 months after initial diagnosis, 3 months after initial diagnosis, 4 months after initial diagnosis, 5 months after initial diagnosis, 6 months after initial diagnosis, 1 year after initial diagnosis, etc. In other embodiments, the efficacy of anti-MAGEB2 immunotherapy against a MAGEB2-expressing tumor may be monitored repeatedly after initiation of anti-MAGEB2 immunotherapy, e.g., 1 month after initiation of treatment, 2 months after initiation of treatment, 3 months after initiation of treatment, 4 months after initiation of treatment, 5 months after initiation of treatment, 6 months after initiation of treatment, 1 year after initiation of treatment, etc.

[0198] Sequence modifications of MAGEB2-binding constructs The term "amino acid" or "amino acid residue" typically refers to amino acids having art-recognized definitions such as amino acids selected from the group consisting of alanine (Ala or A); arginine (Arg or R); asparagine (Asn or N); aspartic acid (Asp or D); cysteine ​​(Cys or C); glutamine (GIn or Q); glutamic acid (GIu or E); glycine (GIy or G); histidine (His or H); isoleucine (Ile or I); leucine (Leu or L); lysine (Lys or K); methionine (Met or M); phenylalanine (Phe or F); proline (Pro or P); serine (Ser or S); threonine (Thr or T); tryptophan (Trp or W); tyrosine (Tyr or Y); and valine (Val or V), although modified, synthetic or rare amino acids may be used as appropriate. There are essentially four different classes of amino acids determined by their different side chains: (1) Non-polar and neutral (uncharged): Ala, Gly, Ile, Leu, Met, Phe, Pro, Val (2) Polar and neutral (uncharged): Asn, Cys (slightly polar), Gln, Ser, Thr, Trp (slightly polar), Tyr (3) Acidic and polar (negatively charged): Asp and Glu (4) Basic and polar (positively charged): Arg, His, Lys. Hydrophobic amino acids can be divided according to whether they have an aliphatic or aromatic side chain. Phe and Trp (very hydrophobic), Tyr and His (less hydrophobic) are classified as aromatic amino acids. Strictly speaking, aliphatic means that the side chain contains only hydrogen and carbon atoms. By this strict definition, amino acids with aliphatic side chains are alanine, isoleucine, leucine (also norleucine), proline and valine. The very short side chain of alanine means that it is not particularly hydrophobic, and proline has an unusual geometry that gives it a special role in proteins. It is often convenient to consider methionine in the same category as isoleucine, leucine and valine, although it also contains a sulfur atom. The unifying theme is that these amino acids contain mainly non-reactive and flexible side chains. The amino acids alanine, cysteine, glycine, proline, serine and threonine are often grouped together because of their small size. Gly and Pro can affect chain orientation.

[0199] Amino acid modifications include, for example, deletion of residues from the amino acid sequence of the binding construct, insertion of residues into the amino acid sequence of the binding construct and / or substitution of residues within the amino acid sequence of the binding construct. Any combination of deletion, insertion and / or substitution may be made to arrive at the final binding construct, provided that the final construct possesses the desired properties, e.g., biological activity of the unmodified parent molecule (e.g., binding to MAGEB2). Amino acid changes may also alter post-translational processes of the binding construct, such as changing the number or position of glycosylation sites.

[0200] For example, in each of the CDRs (depending of course on their respective lengths), 1, 2, 3, 4, 5 or 6 amino acids may be inserted, deleted and / or substituted, while in each of the FRs, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids may be inserted, deleted and / or substituted. Amino acid sequence insertions include N- and / or C-terminal additions of amino acids ranging from, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 residues to more than 10, for example 100 or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Insertional variants of the binding constructs of the invention include fusion of a polypeptide to the N- or C-terminus of the binding construct that increases or extends the serum half-life of the binding construct. Such insertions may also occur within the binding construct, for example between the first and second domains.

[0201] The most favorable sites for amino acid modification, particularly amino acid substitution, include the hypervariable regions of the heavy and / or light chains, particularly the individual CDRs, although modification of the FRs in the heavy and / or light chains is also contemplated. Substitutions can be conservative substitutions as described herein. Preferably, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids can be substituted in the CDRs, while 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 25 amino acids can be substituted in the framework regions (FRs), depending on the length of the CDRs or FRs, respectively. For example, if the CDR sequence comprises 6 amino acids, it is contemplated that 1, 2 or 3 of these amino acids are substituted. Similarly, if the CDR sequence comprises 15 amino acids, it is contemplated that 1, 2, 3, 4, 5 or 6 of these amino acids are substituted.

[0202] A useful method for identifying specific residues or regions within a binding construct that are favorably positioned for mutagenesis is called "alanine scanning mutagenesis" and is described, for example, in Cunningham BC and Wells JA (Science. 1989 Jun 2;244(4908):1081-5). Here, residues or groups of residues within the binding construct are identified (e.g., charged residues such as Arg, His, Lys, Asp and Glu) and replaced with neutral or non-polar amino acids (most preferably alanine or polyalanine) to affect the interaction of each amino acid with the epitope of the target protein. Alanine scanning is a technique used to determine the contribution of specific residues to the stability or function of a given protein. Alanine is used due to its non-bulky and chemically inert methyl functional group, despite mimicking the secondary structure preferences of many other amino acids. Sometimes, bulky amino acids such as valine or leucine can be used when conservation of size of the mutated residue is required. This technique can also be useful in determining whether the side chain of a particular residue plays an important role in biological activity. Alanine scanning is usually accomplished by site-directed mutagenesis or randomly by creating a PCR library. In addition, computational methods have been developed to estimate thermodynamic parameters based on theoretical alanine substitutions. Data can be examined by IR, NMR spectroscopy, mathematical methods, bioassays, etc.

[0203] The amino acid positions that show functional sensitivity to the substitution (e.g., as determined by alanine scanning) can then be refined by introducing further or other variants at or for the substitution site. Thus, the site or region at which the amino acid sequence mutation is introduced is predetermined, but the nature of the mutation itself need not be predetermined. For example, to analyze or optimize the performance of a mutation at a given site, alanine scanning or random mutagenesis can be performed at the target codon or region, and the expressed binding construct variants are screened for the optimal combination of desired activity. Techniques for making substitution mutations at a predetermined site in DNA with a known sequence are well known, such as M13 primer mutagenesis and PCR mutagenesis. Screening of mutants is performed, for example, using assays for antigen (e.g., MAGEB2) binding activity and / or cytotoxicity activity.

[0204] Generally, when amino acids are substituted in one or more or all of the CDRs of the heavy and / or light chain, it is assumed that the resulting "replacement" sequence is at least 60% or 65%, more preferably 70% or 75%, even more preferably 80% or 85%, particularly preferably 90% or 95% identical / homologous to the "original" or "parent" CDR sequence. This means that the degree of identity / homology between the original and the substituted sequence depends on the length of the CDR. For example, a CDR with a total of 5 amino acids and containing one amino acid substitution is 80% identical to the "original" or "parent" CDR sequence, while a CDR with a total of 10 amino acids and containing one amino acid substitution is 90% identical to the "original" or "parent" CDR sequence. Thus, the substituted CDRs of the binding constructs of the invention may have different degrees of identity to their original sequences, for example CDRL1 may have 80% homology, while CDRL3 may have 90% homology. The same considerations apply to the framework regions and throughout the VH and VL regions.

[0205] A "variant CDR" is a CDR that has a particular sequence homology, similarity or identity to a parent CDR of the invention and shares a biological function with the parent CDR, including but not limited to at least 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% of the specificity and / or activity of the parent CDR. Typically, the amino acid homology, similarity or identity between the individual variant CDRs is at least 60% relative to the parent sequences shown herein, more typically at least 65% or 70%, preferably at least 75% or 80%, more preferably at least 85%, 90%, 91%, 92%, 93%, 94%, most preferably 95%, 96%, 97%, 98%, 99% and even increased to approximately 100% homology, similarity or identity. The same is true for "variant VH" and "variant VL". According to one embodiment, the sequence variation within "variant VH" and / or "variant VL" does not extend to the CDRs. The present invention is therefore directed to binding constructs as defined herein comprising VH and VL sequences that have a particular sequence homology (see above) to the particular sequences defined herein ("parental" VH and VL), and wherein the CDR sequences are 100% identical to the particular CDR sequences defined herein ("parental" CDRs).

[0206] Preferred substitutions (or replacements) are conservative substitutions. However, any substitution (including non-conservative substitutions or one or more of the "exemplary substitutions" listed in Table 6 below) is envisaged, as long as the binding construct retains the ability to bind to MAGEB2 and / or the CDR, FR, VH and / or VL sequences have an identity to the extent of at least 60% or 65%, more preferably at least 70% or 75%, even more preferably at least 80% or 85%, particularly preferably at least 90% or 95% with the original or parent sequence.

[0207] Conservative replacements (also called conservative mutations or conservative substitutions) are amino acid replacements that change a given amino acid to a different amino acid with similar biochemical properties (e.g., charge, hydrophobicity, size). Conservative replacements in proteins often have less effect on protein function than non-conservative replacements. Conservative replacements are shown in Table 6. Exemplary conservative replacements are shown as "exemplary replacements". If such replacements result in changes in biological activity, more substantial changes can be introduced, as further described herein with respect to amino acid classes, and the products can be screened for desired properties.

[0208] [Table 10]

[0209] Substantial modification of the biological properties of the binding constructs of the invention is achieved by selecting substitutions that have significantly different effects on (a) the structure of the polypeptide backbone, e.g., sheet or helix structure, in the region of the substitution, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining the bulk of the side chain. Non-conservative substitutions usually involve exchanging a member of one of the above-defined amino acid classes (e.g., polar, neutral, acidic, basic, aliphatic, aromatic, small...) for another class. Any cysteine ​​residue that is not involved in maintaining the proper conformation of the binding construct can generally be substituted with serine to improve the oxidative stability of the binding construct.

[0210] In addition to the above substitutions, other substitutions in CDRs that contribute to binding can be made. For example, the consensus sequences shown in Table 25 herein provide such substitutions. In certain embodiments, deletions guided by the consensus sequences provided can also be made. Those skilled in the art can easily make various amino acid substitutions and their functional equivalents or deletions using the guidance provided herein by the consensus sequences shown in Table 25.

[0211] Sequence identity, homology and / or similarity of amino acid sequences can be determined using standard techniques known in the art, including, but not limited to, the local sequence identity algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, the sequence identity alignment algorithm of Needleman and Wunsch (J Mol Biol. 1970 Mar;48(3):443-53), the similarity search method of Pearson and Lipman (Proc Natl Acad Sci USA. 1988 Apr;85(8):2444-8), computerized implementations of these algorithms (GAP, BESTFIT, FASTA and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis), Devereux et al. (Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387-95) or by visual inspection. Percent identity is assumed to be calculated by FastDB based on the following parameters: mismatch penalty of 1; gap penalty of 1; gap size penalty of 0.33; and joining penalty of 30. See also "Current Methods in Sequence Comparison and Analysis," Macromolecule Sequencing and Synthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R. Liss, Inc.

[0212] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignments to create a multiple sequence alignment from a group of related sequences. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (J Mol Evol. 1987;25(4):351-60), which is similar to the method described by Higgins and Sharp (Comput Appl Biosci. 1989 Apr;5(2):151-3). Useful PILEUP parameters include a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.

[0213] Another example of a useful algorithm is the BLAST algorithm described in Altschul et al. (J Mol Biol. 1990 Oct 5;215(3):403-10.); Altschul et al., (Nucleic Acids Res. 1997 Sep 1;25(17):3389-402) and Karlin and Altschul (Proc Natl Acad Sci USA. 1993 Jun 15;90(12):5873-7). A particularly useful BLAST program is the WU-Blast-2 program, which was obtained from Altschul et al., (Methods Enzymol. 1996;266:460-80). WU-Blast-2 uses several search parameters, most of which are set to the default values. Adjustable parameters are set to the following values: overlap coverage=1, overlap percentage=0.125, word threshold (T)=II. The HSP S and HSP S2 parameters are dynamic values ​​and are established by the program itself depending on the composition of the particular sequence and the composition of the particular database in which the sequence of interest is searched; however, the values ​​can be adjusted to increase sensitivity.

[0214] An additional useful algorithm is Gapped BLAST as reported by Altschul et al. (Nucleic Acids Res. 1997 Sep 1;25(17):3389-402). Gapped BLAST uses the BLOSUM-62 substitution score, the threshold T parameter is set to 9, the two-hit method for triggering ungapped extensions, the charge gap length k is set to a cost of 10+k, Xu is set to 16, and Xg is set to 40 for the database search phase of the algorithm and 67 for the output phase. Gapped alignments are triggered by a score corresponding to approximately 22 bits.

[0215] Nucleotides encoding the binding construct The present invention provides polynucleotides / nucleic acid molecules encoding the polypeptide constructs of the present invention. Nucleic acid molecules are biopolymers made of nucleotides. Polynucleotides are biopolymers composed of 13 or more nucleotide monomers covalently linked in a chain. DNA (such as cDNA) and RNA (such as mRNA) are examples of polynucleotides / nucleic acid molecules with different biological functions. Nucleotides are organic molecules that function as monomers or subunits of nucleic acid molecules such as DNA or RNA. The nucleic acid molecules or polynucleotides of the present invention can be double-stranded or single-stranded, linear or circular. It is envisaged that the nucleic acid molecules or polynucleotides are contained in a vector. Furthermore, it is envisaged that such vectors are contained in a host cell. The host cell is capable of expressing the combined construct, for example after transformation or transfection with the vector or polynucleotide / nucleic acid molecule of the present invention. For this purpose, the polynucleotide or nucleic acid molecule is operably linked to a control sequence.

[0216] The genetic code is a set of rules that translates the information encoded in genetic material (nucleic acids) into proteins. Biological decoding in living cells is carried out by ribosomes, which use tRNA molecules to carry amino acids and read the mRNA three nucleotides at a time, linking them together in the order specified by the mRNA. The code defines how a sequence of triplet nucleotides, called codons, specifies which amino acid is to be added next during protein synthesis. With some exceptions, a three-nucleotide codon in a nucleic acid sequence specifies one amino acid. Because most genes are coded with the exact same code, this particular code is often referred to as the canonical or standard genetic code.

[0217] Codon degeneracy is the redundancy of the genetic code, which is manifested as multiple combinations of three-base pair codons that specify an amino acid. Degeneracy arises due to the large number of codons compared to the codeable amino acids. Codons that code for one amino acid can differ in any of their three positions, but in most cases, this difference is in the second or third position. For example, the codons GAA and GAG both specify glutamic acid and exhibit redundancy, but neither specifies any other amino acid, and therefore exhibit no ambiguity. The genetic code of various organisms may be biased to use one of several codons that code for the same amino acid over others, i.e., one may be found more frequently than would be expected by chance. For example, leucine is specified by six separate codons, some of which are rarely used. Codon usage tables are available that detail the genomic codon usage for most organisms. In recombinant genetic technology, this effect is often exploited by implementing a technique called codon optimization, where polynucleotides are designed that are preferred by the respective host cell (e.g., human, hamster derived, Escherichia coli or Saccharomyces cerevisiae cells) using these codons, for example to increase protein expression. Thus, it is envisaged that the polynucleotides / nucleic acid molecules of the present disclosure are codon optimized.

[0218] Nevertheless, polynucleotides / nucleic acid molecules encoding the binding constructs of the invention may be designed using any codons that encode the desired amino acids.

[0219] In accordance with this specification, the term "percent (%) nucleic acid sequence identity / homology / similarity" with respect to a nucleic acid sequence encoding a binding construct identified herein is defined as the percentage of nucleotide residues in a candidate sequence that are identical to the nucleotide residues in the coding sequence of the binding construct. One method for aligning two sequences and thereby determining their homology uses the BLASTN module of WU-Blast2 set to default parameters with overlap span and overlap fraction set to 1 and 0.125, respectively. Generally, the nucleic acid sequence homology, similarity or identity between the nucleotide sequence encoding each variant CDR and the nucleotide sequence set out herein will be at least 60%, more typically at least 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% and up to approximately 100% increased homology, similarity or identity. Again, the same applies to the nucleic acid sequences encoding the "variant VH" and / or "variant VL".

[0220] In one embodiment, the percentage of identity to human germline of the binding constructs according to the invention or the first and second domains (binding domains) of these binding constructs is 70% or more or 75% or more, more preferably 80% or more or 85% or more, even more preferably 90% or more, most preferably 91% or more, 92% or more, 93% or more, 94% or more, 95% or more or 96% or more. Identity to human antibody germline gene products is considered to be an important feature to reduce the risk that a therapeutic protein will induce an immune response against the drug in patients under treatment. Hwang WY and Foote J. (Methods. 2005 May; 36(1): 3-10) demonstrate that a reduction in the non-human portion of a drug binding construct results in a reduction in the risk of inducing anti-drug antibodies in patients under treatment. Comparison of a comprehensive number of clinically evaluated antibody drugs with their respective immunogenicity data shows that humanization of the variable regions of the antibody / binding construct tends to make the protein less immunogenic (5.1% of patients on average) than antibodies / binding constructs with unchanged non-human variable regions (23.59% of patients on average). Therefore, a higher degree of identity to human sequences is desirable for protein therapeutics in the form of variable region-based binding constructs. For the purpose of determining germline identity, the amino acid sequence can be calculated by aligning the V region of the VL with the amino acid sequences of the human germline segments and J segments (http: / / www2.mrc-lmb.cam.ac.uk / vbase / ) using Vector NTI software and dividing the identical amino acid residues by the total number of amino acid residues of the VL (%). The same can be done for the VH segment (http: / / www2.mrc-lmb.cam.ac.uk / vbase / ), except that the VH CDR3 can be excluded due to its high diversity and lack of existing human germline VH CDR3 alignment partners. Recombinant techniques can then be used to increase sequence identity to human antibody germline genes.

[0221] According to one embodiment, the polynucleotide / nucleic acid molecule of the invention encoding the binding construct of the invention is in the form of one single molecule or in the form of two or more separate molecules. If the binding construct of the invention is a single-stranded binding construct, the polynucleotide / nucleic acid molecule encoding such a construct is most likely also in the form of one single molecule.

[0222] The same is true for vectors containing polynucleotides / nucleic acid molecules of the invention. When the binding construct of the invention is a single-stranded binding construct, a vector may contain the polynucleotides encoding the binding construct in one single location (as one single open reading frame, ORF). A vector may also contain two or more polynucleotides / nucleic acid molecules in separate locations (with individual ORFs), each of which encodes a different component of the binding construct of the invention. It is envisaged that the vectors containing the polynucleotides / nucleic acid molecules of the invention may be in the form of one single vector or two or more separate vectors. In one embodiment, for the purpose of expressing the binding construct in a host cell, the host cell of the invention should contain the polynucleotides / nucleic acid molecules encoding the binding construct or a vector containing such polynucleotides / nucleic acid molecules in their entirety, meaning that all components of the binding construct (whether encoded as a single molecule or encoded in separate molecules / locations) are post-translationally assembled together to form the biologically active binding construct of the invention.

[0223] The present invention also provides vectors comprising the polynucleotide / nucleic acid molecules of the present invention. A vector is a nucleic acid molecule that is usually used as a vehicle to transfer (foreign) genetic material into a cell for replication and / or expression. The term "vector" includes, but is not limited to, plasmids, viruses, cosmids and artificial chromosomes. Some vectors are specifically designed for cloning (cloning vectors), others for protein expression (expression vectors). So-called transcription vectors are mainly used to amplify their insert. DNA manipulations are usually performed on E. coli vectors, which contain the elements necessary for their maintenance in E. coli. However, vectors can also have elements that allow them to be maintained in another organism, such as yeast, plant or mammalian cells, and these vectors are called shuttle vectors.

[0224] The insertion of the vector into the target or host cell is commonly referred to as transformation for bacterial cells, transfection for eukaryotic cells, and, in addition, the insertion of viral vectors is often referred to as transduction.

[0225] Generally, engineered vectors contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is a nucleotide sequence, usually a DNA sequence, that generally contains an insert (transgene) and a larger sequence that serves as the "backbone" of the vector. The genetic code determines the polypeptide sequence of a given coding region, but other genomic regions can influence when and where these polypeptides are produced. Thus, in addition to the transgene insert and backbone, modern vectors can include the following additional features: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags.

[0226] Vectors, called expression vectors (expression constructs), are specifically intended for the expression of a transgene in a target cell and generally contain regulatory sequences.

[0227] The term "control sequence" refers to a DNA sequence necessary for the expression of an operably linked coding sequence in a particular host organism. For example, control sequences suitable for prokaryotes include promoters, optionally including operator sequences and ribosome binding sites. Eukaryotic cells are known to utilize promoters, polyadenylation signals, Kozak sequences, and enhancers.

[0228] A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA of a presequence or secretory leader is operably linked to DNA of a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a ribosome binding site is operably linked to a coding sequence if it is positioned such that the ribosome binding site promotes translation. Generally, "operably linked" means that the nucleotide sequences being linked are contiguous, and in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0229] "Transfection" is the process of deliberately introducing a nucleic acid molecule or polynucleotide (including vectors) into a target cell. The term is primarily used for non-viral methods in eukaryotic cells. Transduction is often used to describe the virus-mediated transfer of a nucleic acid molecule or polynucleotide. Transfection of animal cells typically involves opening a transient pore or "hole" in the cell membrane to allow uptake of material. Transfection can be performed using biological particles (e.g., viral transfection, also called viral transduction), chemical-based methods (e.g., using calcium phosphate, lipofection, Fugene, cationic polymers, nanoparticles) or physical treatments (e.g., electroporation, microinjection, gene gun, cell squeezing, magnetofection, hydrostatic pressure, impalefection, sonication, optical transfection, heat shock).

[0230] The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides (including vectors) into bacteria, as well as non-animal eukaryotic cells, such as plant cells. Transformation is thus the genetic modification of bacteria or non-animal eukaryotic cells resulting from the direct uptake through the cell membrane from its surroundings and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can occur by artificial means. For transformation to occur, the cells or bacteria must be in a state of transformation competent, which can occur as a time-limited response to environmental conditions such as starvation and cell density, or can occur artificially.

[0231] Furthermore, the present invention provides a host cell transformed or transfected with a polynucleotide / nucleic acid molecule of the invention or a vector of the invention.

[0232] As used herein, the term "host cell" or "recipient cell" is intended to include any individual cell or cell culture that can be or has been a recipient of a vector, an exogenous nucleic acid molecule and / or a polynucleotide encoding a binding construct of the present invention, and / or a recipient of the binding construct itself. Introduction of an individual material into a cell is accomplished by transformation, transfection, etc. (see above). The term "host cell" is also intended to include the passage or potential succession of a single cell. Since certain modifications may occur in subsequent generations, either due to natural, accidental or deliberate mutations or due to environmental influences, such progeny may not in fact be completely identical (in morphology or genomic or total DNA complement) to the parent cell, but still be within the scope of the term as used herein. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria (such as E. coli), yeast cells, fungal cells, plant cells, and animal cells (e.g., insect cells and mammalian cells, such as hamster, mouse, rat, macaque, or human).

[0233] In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the conjugation constructs of the present invention. Saccharomyces cerevisiae, or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms. However, Kluyveromyces hosts such as Schizosaccharomyces pombe, K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans and K. marxianus; Yarrowia (EP 402226); Pichia pastoris; pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts (such as A. nidulans and A. niger), many other genera, species, and strains are publicly available and useful herein.

[0234] Suitable host cells for the expression of the glycosylation binding construct are derived from multicellular organisms. Examples of invertebrate cells include plant cells and insect cells. Many strains and variants of baculoviruses and corresponding permissive insect host cells from hosts (e.g., Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly) and Bombyx mori) have been identified. Various virus strains for transfection are publicly available, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the viruses herein according to the invention, particularly for transfection of Spodoptera frugiperda cells.

[0235] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis and tobacco can also be used as hosts. Cloning and expression vectors useful for producing proteins in plant cell cultures are known to those skilled in the art. See, for example, Hiatt et al., Nature (1989) 342:76-78; Owen et al. (1992) Bio / Technology 10:790-794; Artsaenko et al. (1995) The Plant J 8:745-750 and Fecker et al. (1996) Plant Mol Biol 32:979-986.

[0236] However, most interest has been in vertebrate cells, and propagation of vertebrate cells in culture (cell culture) has become a routine procedure. Examples of useful mammalian host cell lines include SV40 transformed monkey kidney CV1 lines (e.g., COS-7, ATCC CRL 1651); human embryonic kidney lines (e.g., 293 cells or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (e.g., BHK, ATCC CCL 10); Chinese hamster ovary cells / -DHFR (e.g., CHO, Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (e.g., TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVI ATCC CCL 70, etc.); African green monkey kidney cells (VERO-76, ATCC CRL1587, etc.); human cervical carcinoma cells (HELA, ATCC CCL 2, etc.); canine kidney cells (MDCK, ATCC CCL 34, etc.), buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442, etc.); human lung cells (W138, ATCC CCL 75, etc.); human hepatocytes (Hep G2, 1413 8065, etc.); mouse mammary tumor (MMT 060562, ATCC CCL-51, etc.); TRI cells (Mather et al., Annals NY Acad. Sci. (1982) 383:44-68); MRC 5 cells; FS4 cells; and human hepatoma lines (Hep G2, etc.).

[0237] Preparation of binding constructs In a further embodiment, the invention provides a method of producing a binding construct of the invention, said method comprising culturing a host cell of the invention under conditions permissive for expression of the binding construct of the invention and recovering the produced binding construct from the culture.

[0238] As used herein, the term "culture" refers to the maintenance, differentiation, growth, proliferation and / or propagation of cells in vitro under suitable conditions in a medium. Cells are grown and maintained in a cell growth medium at an appropriate temperature and gas mixture. Culture conditions vary widely for different cell types. Typical growth conditions are a temperature of about 37°C, a CO2 concentration of about 5% and a humidity of about 95%. Growth medium recipes can vary, for example, in pH, concentration of carbon source (e.g., glucose), nature and concentration of growth factors and the presence of other nutrients (e.g., amino acids or vitamins). Growth factors used in supplemented media are often derived from serum of animal blood (e.g., fetal bovine serum (FBS), calf serum (FCS), horse serum and porcine serum). Cells can be grown either in suspension culture or as adherent cultures. Some cell lines have been modified to be viable in suspension culture and can therefore grow to higher densities than would be possible in adherent conditions.

[0239] The term "expression" includes all steps involved in the production of the binding construct of the present invention, including, but not limited to, transcription, post-transcriptional modification, translation, folding, post-translational modification, targeting to a specific intracellular or extracellular location, and secretion. The term "recovering" refers to a series of processes intended to isolate the binding construct from a cell culture. The "recovery" or "purification" process separates the protein and non-protein portions of the cell culture, and can ultimately separate the desired binding construct from all other polypeptides and proteins. The separation steps usually exploit differences in protein size, physicochemical properties, binding affinity, and biological activity. Preparative purification aims to produce relatively large amounts of purified protein for subsequent use, while analytical purification produces relatively small amounts of protein for various research or analytical purposes.

[0240] When using recombinant techniques, the binding constructs can be produced intracellularly in the periplasmic space or directly secreted into the medium. If the binding constructs are produced intracellularly, the first step is to remove particulate debris of host cells or lysed fragments, for example by centrifugation or ultrafiltration. The binding constructs of the invention can be produced in bacteria, such as E. coli. After expression, the constructs can be isolated in a soluble fraction from the bacterial cell paste and purified, for example by affinity chromatography and / or size exclusion. Final purification can be performed in a manner similar to the process for purifying binding constructs expressed in mammalian cells and secreted into the medium. Carter et al. (Biotechnology (NY) 1992 Feb;10(2):163-7) describe a procedure for isolating antibodies secreted into the periplasmic space of E. coli.

[0241] Where the antibody is secreted into the medium, supernatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, e.g., an ultrafiltration unit.

[0242] The binding constructs of the invention prepared from the host cells can be recovered or purified using, for example, hydroxylapatite chromatography, gel electrophoresis, dialysis and affinity chromatography. Other techniques for protein purification, such as fractionation on ion exchange columns, mixed mode ion exchange, HIC, ethanol precipitation, size exclusion chromatography, reversed-phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on anion or cation exchange resins (such as polyaspartic acid columns), immunoaffinity (such as protein A / G / L) chromatography, chromatofocusing, SDS-PAGE, ultracentrifugation and ammonium sulfate precipitation, are also available depending on the binding construct to be recovered.

[0243] Protease inhibitors may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of contaminants.

[0244] formulation Furthermore, the present invention provides a composition or formulation comprising a binding construct of the invention or a binding construct produced by a process of the invention.

[0245] Compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

[0246] The composition may include a pharmaceutically acceptable carrier. In general, as used herein, "pharmaceutically acceptable carrier" refers to any and all aqueous and non-aqueous solutions, sterile solutions, solvents, buffer solutions, such as phosphate buffered saline (PBS) solutions, water, suspensions, emulsions such as oil / water emulsions, various wetting agents, liposomes, dispersion media and coatings that are compatible with drug administration, particularly parenteral administration. The use of such media and drugs in pharmaceutical compositions is well known in the art, and compositions containing such carriers can be formulated by well-known conventional methods.

[0247] Certain embodiments provide pharmaceutical compositions comprising a binding construct of the invention and further one or more excipients, such as those exemplarily described in this section and elsewhere herein. In the present invention, excipients may be used for a variety of purposes, such as adjusting the physical, chemical or biological properties of the formulation (e.g., adjusting the viscosity) and / or may be used in the processes of the invention to improve efficacy and / or stabilize such formulations and processes against degradation and deterioration due to, for example, manufacturing, shipping, storage, preparation before use, administration and subsequent stresses. Excipients should generally be used at their lowest effective concentration.

[0248] In certain embodiments, the compositions may contain formulation materials for the purpose of modifying, maintaining or preserving certain properties of the composition, such as pH, osmolality, viscosity, clarity, color, isotonicity, odor, sterility, stability, dissolution or release rate, adsorption or penetration, etc. (Remington's Pharmaceutical Sciences, 18th Edition, 1990, Mack Publishing (See, Company, 2006, Foreword). In such embodiments, suitable formulation materials may include, but are not limited to, amino acids, antimicrobial agents such as antibacterial and antifungal agents, antioxidants, buffers, buffer systems and buffering agents (which are used to maintain the composition at or slightly below physiological pH, typically within a pH range of about 5 to about 8 or 9), non-aqueous solvents, vegetable oils and injectable organic esters, water, aqueous carriers including alcohol / aqueous solutions, emulsions or suspensions (including saline and buffered media), biodegradable polymers (such as polyesters), bulking agents, chelating agents, isotonicity and absorption retarding agents, complexing agents, fillers, carbohydrates, (low molecular weight) proteins, polypeptides or protein carriers (preferably of human origin), colorants and flavoring agents, sulfur-containing reducing agents, diluents, emulsifiers, hydrophilic polymers, salt-forming counterions, preservatives, metal complexes, solvents and co-solvents, sugars and sugar alcohols, suspending agents, surfactants or wetting agents, stability enhancers, isotonicity enhancers, parenteral or intravenous delivery vehicles.

[0249] Various components of the composition may have different effects, for example, amino acids may act as buffers, stabilizers and / or antioxidants, mannitol may act as a bulking agent and / or tonicity enhancer, sodium chloride may act as a delivery vehicle and / or tonicity enhancer, etc.

[0250] In certain embodiments of the invention, salts may be used, for example, to adjust the ionic strength and / or tonicity of a composition or formulation and / or to improve the solubility and / or physical stability of the binding construct or other components of the composition of the invention. Ions can stabilize proteins in their native state by binding to charged residues on the surface of the protein and by shielding the charged and polar groups of the protein, reducing the strength of its electrostatic, attractive and repulsive interactions. Ions can also stabilize proteins in their denatured state, particularly by binding to the denatured peptide bonds (--CONH) of the protein. Furthermore, ionic interactions with charged and polar groups in proteins can also reduce intermolecular electrostatic interactions, thereby preventing or reducing protein aggregation and insolubilization.

[0251] Ionic species vary significantly in their effects on proteins. Several taxonomic rankings of ions and their effects on proteins have been developed that can be used in formulating pharmaceutical compositions according to the invention. One example is the Hofmeister series, which ranks ionic and polar non-ionic solutes by their effect on the conformational stability of proteins in solution. Stabilizing solutes are termed "kosmotropic". Destabilizing solutes are termed "chaotropic". Cosmotropes are commonly used in high concentrations to precipitate proteins from solution ("salting out"). Chaotropes are commonly used to denature and / or solubilize proteins ("salting in"). The relative effects of an ion on "salting in" and "salting out" define the ion's position in the Hofmeister series.

[0252] Free amino acids can be used in formulations or compositions containing the binding constructs of the present invention according to various embodiments of the present invention as bulking agents, stabilizers and antioxidants and for other standard uses. Certain amino acids can be used to stabilize proteins in the formulation, while others are useful during lyophilization to ensure the correct cake structure and properties of the active ingredient. Some amino acids can be useful to inhibit protein aggregation in both liquid and lyophilized formulations, while others are useful as antioxidants.

[0253] Polyols are cosmotropic and are useful as stabilizers in both liquid and lyophilized formulations to protect proteins from physical and chemical degradation processes. Polyols are useful to adjust the tonicity of the formulation and to protect against freeze-thaw stress during transportation or for bulk preparation during manufacturing processes. Polyols can also function as cryoprotectants in the context of the present invention.

[0254] Certain embodiments of the formulation or composition comprising the binding construct of the present invention can include surfactants. Proteins are prone to surface adsorption, denaturation, and consequent aggregation at air-liquid, solid-liquid, and liquid-liquid interfaces. These adverse interactions generally scale inversely with protein concentration and are typically exacerbated by physical agitation, such as those that occur during product shipping and handling. Surfactants are routinely used to prevent, minimize, or reduce surface adsorption.

[0255] Detergents are also commonly used to control the conformational stability of proteins. In this regard, the use of detergents is protein specific, since one particular detergent will typically stabilize some proteins and destabilize others.

[0256] Certain embodiments of the formulation or composition comprising the binding construct of the present invention may include one or more antioxidants. The harmful oxidation of proteins in pharmaceutical formulations can be prevented to some extent by maintaining appropriate levels of ambient oxygen and temperature and avoiding exposure to light. Antioxidant excipients can also be used to prevent oxidative degradation of proteins. It is envisioned that antioxidants for use in therapeutic protein formulations according to the present invention are water-soluble and can maintain their activity throughout the shelf life of the product (composition comprising the binding construct).

[0257] Antioxidants can also damage proteins and should therefore be selected specifically to eliminate or sufficiently reduce the possibility of the antioxidant damaging the binding construct or other proteins in the formulation.

[0258] The formulations according to the invention may contain metal ions, which are cofactors for proteins and are required to form protein coordination compounds, such as zinc, which is required to form certain insulin suspensions. Metal ions can also inhibit some processes that break down proteins. However, metal ions also catalyze physical and chemical processes that break down proteins. Magnesium ions (10-120 mM) can be used to inhibit the isomerization of aspartic acid to isoaspartic acid. Ca +2 ions (up to 100 mM) can increase the stability of human deoxyribonuclease. +2 , Mn +2 and Zn +2 can destabilize rhDNase. +2 and Sr +2 can stabilize factor VIII, which is +2 , Mn +2 and Zn +2 , Cu +2 and Fe +2 The aggregation can be destabilized by Al +3 It can be increased by ions.

[0259] Certain embodiments of the formulations or compositions comprising the binding constructs of the present invention may include one or more preservatives. Preservatives are necessary, for example, during the development of multi-dose parenteral formulations involving more than one withdrawal from the same container. Their main function is to inhibit microbial growth and ensure sterility of the product over the shelf life or use period of the formulation. Although preservatives have a long history of use in small molecule parenteral substances, developing protein formulations containing preservatives can be a challenge. Preservatives quite often have a destabilizing effect on proteins (aggregation), which is the main reason for their limited use in multi-dose protein formulations. During the formulation and development of preserved dosage forms, several aspects must be considered. The effective preservative concentration in the product must be optimized. This requires testing a given preservative in the dosage form for a concentration range that confers antimicrobial efficacy without compromising protein stability.

[0260] The development of liquid formulations containing preservatives is more challenging than lyophilized formulations. Lyophilized products can be lyophilized without preservatives and reconstituted with a diluent containing the preservative at the time of use. This reduces the time that the preservative is in contact with the binding construct, greatly reducing the associated stability risks. In liquid formulations, the effectiveness and stability of the preservative must be maintained throughout the entire shelf life of the product. An important point to note is that the effectiveness of the preservative should be demonstrated in the final formulation containing the active drug and all excipient components. Once the pharmaceutical composition is formulated, it can be stored in a sterile vial as a solution, suspension, gel, emulsion, solid, crystal, or as a dehydrated or lyophilized powder. Such formulations can be stored in a form ready for use or in a form that is reconstituted before administration (e.g., lyophilized).

[0261] kit In a further embodiment, the invention provides a kit comprising a binding construct that binds to MAGEB2 and is useful for detecting MAGEB2 in a tumor sample, the binding construct comprising any of the binding constructs disclosed herein. In one embodiment, the binding construct useful for detecting MAGEB2 is a monoclonal antibody that binds to MAGEB2. In a further embodiment, the kit further comprises a therapeutic agent effective to treat a cancer expressing MAGEB2. In certain embodiments, the kit may optionally be provided with a package insert comprising instructions for detecting MAGEB2 and administering the cancer therapeutic agent.

[0262] In a further embodiment, the kit further comprises binding constructs (eg antibodies) useful for detecting various biomarkers other than MAGEB2, for example further comprising a MAGEA4 binding construct or a MAGEA8 binding construct.

[0263] As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, reference to a "reagent" includes one or more of such various reagents, and reference to a "method" includes reference to equivalent steps and methods known to those of skill in the art that may be modified for or substituted for the method described herein.

[0264] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0265] The term "and / or", wherever used in this specification, includes the meanings of "and", "or" and "any other combination of the elements connected by the term".

[0266] As used herein, the term "about" or "approximately" means within ±20%, preferably within ±15%, more preferably within ±10%, and most preferably within ±5% of a given value or range, including specific values, e.g., "about 50" includes the value "50."

[0267] Throughout this specification and claims, unless the context otherwise requires, "comprise" and variations such as "comprises" and "comprising" will be understood to imply the inclusion of a stated integer or step or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. As used herein, the term "comprise" may be substituted with the term "containing" or "including," or, as used herein, may sometimes be substituted with the term "having."

[0268] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0269] In each instance herein, any of "comprising," "consisting essentially of," and "consisting of" may be replaced with either of the other two terms.

[0270] The above description and the following examples provide exemplary configurations, however, it should be understood that the invention is not limited to the particular methodology, techniques, protocols, materials, reagents, substances, etc. described herein, as such may vary. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined solely by the claims. Aspects of the invention are provided in the independent claims. Some optional features of the invention are provided in the dependent claims.

[0271] All publications and patents cited throughout the text of this specification, whether supra or infra, including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc., are hereby incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate any disclosure by virtue of prior invention. To the extent that material incorporated by reference contradicts or is inconsistent with this specification, the present specification takes precedence over any such material.

[0272] A better understanding of the present invention and its advantages will be obtained from the following examples, which are provided for illustrative purposes only and are not intended, and should not be construed, as limiting the scope of the invention in any way. EXAMPLES

[0273] Example 1: MAGEB2 immunogen design Protein sequences of the following 35 H. sapiens MAGE family members were downloaded from UniProtKB (www.uniprot.org): MAGEA1 (P43355), MAGEA2 (P43356), MAGEA3 (P43357), MAGEA4 (P43358), MAGEA5 (P43359), MAGEA6 (P43360), MAGEA8 (P43361), MAGEA9 (P43362), MAGEA10 (P43363), MAGEA11 (P43364), MAGEA12 (P43365), MAGEB1 (P43366), MAGEB2 (O15479), MAGEB3 (O15479), MAGEB4 (O15479), MAGEB5 (O15479), MAGEB6 (O15479), MAGEB7 (O15479), MAGEB8 (O15479), MAGEB9 (O15479), MAGEB10 (P43360), MAGEB11 (P43361), MAGEB12 (P43362), MAGEB13 (P43363), MAGEB14 (P43364), MAGEB15 (P43365), MAGEB16 (P43366), MAGEB17 (P43366), MAGEB18 (P43366), MAGEB19 (P43366), MAGEB20 (P43366), MAGEB21 (P43366), MAGEB22 (P43366), MAGEB23 (P43366), MAGEB24 (P43366), MAGEB25 (P43366), MAGEB26 (P43366), MAGEB27 (P43366), 5481), MAGEB5(Q9BZ81), MAGEB6(Q8N7X4), MAGEB10(Q96LZ2), MAGEB16(A2A368), MAGEB17( A8MXT2), MAGEB18(Q96M61), MAGEC1(O60732), MAGEC2(Q9UBF10), MAGEC3(Q8TD91), MAGED1( Q9Y5V3), MAGED2 (Q9UNF1), MAGED4 (Q96JG8), MAGEE1 (Q9HCI5), MAGEE2 (Q8TD90), MAGEH1 (Q9H213), MAGEF1 (Q9HAY2), MAGEL2 (Q9UJ55), NSE3 (Q96MG7), TROP (Q12816) and NECD (Q99608). Clades were determined using the Neighbor-Joining Tree Builder algorithm in Geneious v10.2.

[0274] Shannon entropy, a quantitative metric that represents the amino acid diversity observed at a given position in an alignment, was used to select MAGEB2 peptide immunogens with low amino acid conservation across the entire MAGE family and MAGE-B clades (B1, B2, B3, B4, B5, B17). MAGE family or MAGE-B clade members were aligned using MUSCLE (RCEdgar. Nucleic Acids Res. 2004;32(5),1792-1797). Shannon entropy was calculated for each position in the alignment as H S (j)=-Σi f ij log2f ij (where HS(j) is the Shannon entropy at position j and fij is the frequency of AAi at position j) Shannon entropy was calculated using the formula: (1) where HS(j) is the Shannon entropy at position j and fij is the frequency of AAi at position j. Shannon entropy was mapped to the structure of the MAGE homology domain (MHD) of MAGEA4 (PDB ID 2wa0) or the Rosetta homology model of the MHD of MAGEB2 using a custom script that replaced the PDB b-factor of each atom with the normalized Shannon entropy value for its corresponding residue, and then visualized in PyMOL v1.8 (PyMOL Molecular Graphics System, Version 1.8 Schroedinger, LLC) using color by b-factor.

[0275] A custom script was used to conclusively rank regions of length n (n = 5-40 amino acids) within the alignment from highest to lowest average Shannon entropy (least to most conserved) by a sliding window method. Across the MAGE family, regions of length 10-30 with the lowest conservation across the family cluster into three distinct regions; namely, two regions on the N-terminal side of the MHD and one region within the N-terminal helix of the MHD. Analysis of the MAGE-B clade revealed a clade-specific region of low conservation located on a loop at the interface of the two globular lobes of the MHD. The MAGEB2 peptide immunogen was selected from the following three identified regions of low conservation: the N-terminal region of the MHD, the N-terminal helix of the MHD and the MDH interface loop region. Peptide coverage within these regions was refined based on consideration of structural features such as average Shannon entropy scores, visual inspection of sequence alignments, solvent accessibility and conservation of secondary structure as isolated peptides predicted by PSIPRED (Jones DT. (1999) Protein secondary structure prediction based on position-specific scoring matrices. J. Mol. Biol. 292:195-202) and whether a sequence blast against the non-reduced H. sapiens proteome revealed hits with high homology in non-MAGE proteins. The regions of MAGEB2 selected as peptide immunogens were: MAGEB2a.a.43-76: SSVSGGAASSSPAAGIPQEPQRAPTTAAAAAAGV (N-terminal region peptide); MAGEB2a.a.95-125: SSSQASTSTKSPSEDPLTRKSGSLVQFLLYK (N-terminal helix peptide of MHD); and MAGEB2a.a.185-200: DLTDEESLLSSWDFPR (mid-loop peptide of MHD).

[0276] Peptides from MAGE homologs with >50% pairwise identity to the MAGEB2 immunogen were selected for experimental counterscreening. Additional MAGEB2 homolog peptides were selected for counterscreening. For MAGEB2a.a.43-76 (N-terminal region peptides), the counterscreening peptides selected were derived from MAGE-B1, B4, B17, A4, A1, A5, A12, A6, A3, A2, A11, and A8. For MAGEB2a.a.95-125 (N-terminal helical peptides of MHD), the counterscreening peptides selected were derived from MAGE-B4, B5, B17, C3, A11, A8, A9, A3, A10, F1, A4, A5, A12, C2, A2, E1, and C1. For MAGEB2a.a.185-200 (mid-loop peptide of MHD), a single counterscreening peptide derived from MAGEB1 was selected.

[0277] Example 2: Antibody Generation and Selection immunization Rabbits were immunized according to standard protocols and splenocytes were isolated and frozen from immunized animals.

[0278] Preparation of peptides for antigen-specific selection The 27 biotin-labeled MAGE counterscreening peptides (Table 7) were pooled together at equimolar concentrations to make a working cocktail. The cocktail was conjugated to neutravidin (Fisher:PI31000) at a molar ratio of 4:1 (neutravidin:peptide) in 2% FBS FACS buffer (calcium- and magnesium-free PBS with 2 v / v% FBS). The mixture was incubated at 4° C. for 15 hours. Each of the three biotin-labeled MAGEB2 target peptides (N-terminal region peptide, mid-loop region peptide, N-terminal helix peptide, see Table 8) was conjugated to different fluorescently tagged streptavidin using the same 4:1 (streptavidin:peptide) molar ratio in 2% FBS FACS buffer and then incubated at 4° C. for 15 minutes. The MAGEB2 N-terminal peptide was conjugated to BV421 streptavidin (BD:563259), the MAGEB2 mid-loop region peptide was conjugated to BV605 streptavidin (BD:563855) and the MAGEB2 N-terminal helix peptide was conjugated to Alexa Fluor 647 streptavidin (Jackson:016-600-084).

[0279] Unbound streptavidin or neutravidin was quenched by adding an excess of D-biotin (Fisher:BP232-1) at a molar ratio of 600:1 (D-biotin:streptavidin or neutravidin) and the peptides were then incubated for an additional 15 min at 4° C. The three MAGEB2 target peptides were pooled together immediately before use.

[0280] [Table 11]

[0281] [Table 12]

[0282] Preparation of rabbit splenocytes for antigen-specific selection Rabbit splenocytes were thawed in 10% ICM (immune cell culture medium) and dead cells were removed using Miltenyi Biotec MACS dead cell removal kit (Miltenyi: 130-090-101). Cells were then washed in 10 mL of cold 2% FBS FACS buffer. After centrifugation at 400G for 4 min, the supernatant was removed using a serological pipette and a cocktail of neutravidin-conjugated MAGE counterscreening peptides was added. Cells were incubated at 4°C for 30 min, after which three streptavidin-conjugated MAGEB2 target peptides were added along with 5 μg of FITC-conjugated goat anti-rabbit IgG Fc antibody (Serotec: STAR121F) to stain IgG-expressing antigen-specific cells. The final concentrations of peptides in a 1 mL reaction volume were 120 nM for the cocktail of counterscreening peptides and 16.5 nM for each target peptide. The cells were incubated for an additional 30 min at 4° C., then washed in 10 mL of cold 2% FBS FACS buffer and centrifuged again. After removing the supernatant, the cells were resuspended in 2 mL of 10% ICM and then passed through a 40 μm cell strainer to remove clumps. 10 μL of 7-AAD viability stain (Fisher: 00-6993-50) was added to the cells 10 min before sorting.

[0283] Selection and culture of antigen-specific cells Cells were sorted on a BD FACSAria III equipped with a 405 nm laser (detection: 450 / 50 nm, 610 / 20 nm), a 488 nm laser (detection: 530 / 30 nm, 695 / 40 nm) and a 640 nm laser (detection: 730 / 45 nm) using a 100 micron nozzle and 20 Psi sheath pressure. Dead cells were excluded by gating on the population not fluorescent in 7-AAD and MAGEB2 target peptide binders were selected by drawing three gates with the following selection criteria: FITC+ / BV421+, FITC+ / BV605+, FITC+ / Alexa Fluor 647+ (see Figure 1). The first sorting was performed using yield precision mode to enrich the target population from ~0.1% to ~50%, followed by a second round of sorting using purity precision mode to single cell sort MAGEB2 target peptide binders into 384-well tissue culture plates pre-loaded with 10% ICM supplemented with 4% rabbit T cell supernatant, 2 μL / mL of anti-rabbit IgG microbeads (Miltenyi: 130-048-602) and gamma-irradiated EL4 cells at a density of 437,500 cells / mL. Plates were kept in a tissue culture incubator (37 °C, 5% CO2) and after 7 days growth was assessed by visual scoring using an inverted microscope. After 8 days, supernatants were harvested from tissue culture plates and cells were lysed in buffer RLT (Qiagen: 79216) and frozen at -80 °C.

[0284] Early detection of MAGEB2 antibodies B cell culture supernatants were screened for the presence of positive secreted antibodies using a rabbit IgG capture ELISA. Goat anti-rabbit IgG (Fc) (Jackson, Cat. No. 111-005-046) was coated onto Corning 3702 384-well polystyrene plates in 1× phosphate buffered saline (PBS) overnight at 4° C.

[0285] The plates were washed for 4 cycles using a Titertek plate washer and blocked with 1x PBS / 1% milk. The plates were then aspirated and the B cell culture supernatants were finally diluted 1:5 in 1x PBS / 1% milk and incubated for 1 hour at room temperature. The plates were washed for 4 cycles and then horseradish peroxidase (HRP)-conjugated goat anti-human IgG (Fc) (Jackson, Cat. No. 111-035-046) prepared in 1x PBS / 1% milk was added and incubated for 1 hour at room temperature. The plates were washed for 4 cycles and then 1-step TMB (Neogen Cat. No. 319177) was added for 30 minutes at room temperature followed by quenching with 1N HCl. The plates were read at OD450nm on a Multiscan Ascent plate reader and a total of 129 rabbit antibodies were positive with a breakdown of the number of hits per region shown in Table 9 below.

[0286] [Table 13]

[0287] Identification of MAGEB2-specific antibodies Binding of MAGEB2 antibodies was tested using biotinylated MAGEB2 peptides coated onto xMAP LumAvidin microspheres or Luminex beads (Luminex catalog number L100-LXXX-01). These avidin-conjugated microparticles are color-coded into distinct spectral regions, allowing non-covalent binding of biotinylated proteins with unique combinations of fluorescent dyes. To validate the rabbit IgG capture ELISA results in a first bead-based FACS assay, a panel of 129 rabbit antibodies was tested against the same three peptides with which the rabbits were immunized: MAGEB2 mid-loop region peptide, MAGEB2 N-terminal helix peptide, and MAGEB2 N-terminal region peptide. The three biotinylated peptides were coated with three different LumAvidin beads in FACS buffer [1x PBS + 2% fetal bovine serum (Hyclone catalog number SH30396.03)] for 30 minutes at room temperature in the dark. The beads were then washed twice in FACS buffer by centrifugation at 3500 RPM for 2 minutes. The coated beads were resuspended, pooled together (3 portions) and added to B cell culture supernatants on Corning 3897 96-well V-bottom polystyrene plates at a final dilution of 1:2 and incubated at room temperature in the dark for 1 hour. After two further washes in FACS buffer, Alexa 488-conjugated goat anti-rabbit IgG (Fc) (Jackson, Cat. No. 111-545-046) made in FACS buffer was added and incubated at room temperature in the dark for 15 minutes. After the final wash, the plates were read on an iQue FACS machine with an Intellicyte autosampler, flow cytometry measurements were obtained according to the manufacturer's recommendations, and data analysis was performed with Intellicyt ForeCyt® Enterprise Client Edition 6.2 (R3). A total of 82 wells were identified as MAGEB2 positive, with a breakdown of the number of hits per region as shown in Table 10 below.

[0288] [Table 14]

[0289] In a second bead-based FACS assay, a panel of 129 rabbit antibodies was tested against nine other peptides of similar sequence to determine MAGEB2 specificity: MAGEB1 mid-loop region peptide, MAGEB4 N-terminal helix peptide, MAGEB5 N-terminal helix peptide, MAGE17 N-terminal helix peptide, MAGEF1 N-terminal helix peptide, MAGEE1 N-terminal helix peptide; MAGEB1 N-terminal peptide, MAGEB4 N-terminal region peptide, and MAGEB17 N-terminal region peptide. The nine biotinylated peptides were coated with nine different LumAvidin beads in FACS buffer [1x PBS + 2% fetal bovine serum (FBS)] for 30 minutes at room temperature in the dark. The beads were then washed twice in FACS buffer by centrifugation at 3500 RPM for 2 minutes. The coated beads were resuspended, pooled together (9 portions) and added to rabbit B cell culture supernatants on Corning 3897 96-well V-bottom polystyrene plates at a final dilution of 1:2 and incubated at room temperature for 1 hour in the dark. After two further washes in FACS buffer, Alexa 488-conjugated goat anti-rabbit IgG (Fc) (Jackson, Cat. No. 111-545-046) made in FACS buffer was added and incubated at room temperature for 15 minutes in the dark. After a final wash, the plates were read on an iQue FACS machine with an Intellicyte autosampler, flow cytometry measurements were obtained according to the manufacturer's recommendations, and data analysis was performed with Intellicyt ForeCyt® Enterprise Client Edition 6.2 (R3). A total of 43 wells were identified as MAGEB2-specific, with a breakdown of the number of hits per region as shown in Table 11 below. Heavy and light chain sequencing was performed on all 43 wells.

[0290] [Table 15]

[0291] Identification of high-affinity MAGEB2 antibodies To determine which of the 43 MAGEB2-specific antibodies to proceed with cloning, we performed limited antigen (LA) kinetic FACS assays with LumAvidin beads using full-length MAGEB2 protein to rank the panel and select the antibodies with the highest affinity.

[0292] huMAGEB2(1-319) in pET23 was transformed into strain BL21(DE)-star pLysS (Invitrogen). A single colony was used to inoculate 2 mL of Luria Broth supplemented with carbenicillin (50 μg / mL) and chloramphenicol (34 μg / mL) and grown with shaking at 37°C for several hours until turbid. Two hundred microliters of the culture was used to inoculate 400 mL of Luria Broth (Teknova) supplemented with selection antibiotic and antifoam 204 (Sigma). These cultures were grown with shaking at 24°C overnight. The turbid culture was used to grow 1 liter of Terrific Broth (Teknova) (in a 4 L baffled shake flask) supplemented with 1% (w / v) glucose, 2 mM MgSO4, 17 μg / L carbenicillin, 12 μg / L chloramphenicol, and antifoam 204 at OD 30. 600 These cultures were inoculated to an OD of 0.2. 600 Grow at 37°C with shaking until OD = 1.0, then shift to 16°C and 600 = 2.0, at which point protein expression was induced with 100 μM IPTG for 16 hours, and cells were pelleted by centrifugation and stored at -80°C until processing.

[0293] The MAGEB2 pellet was resuspended in lysis buffer (100 mM Tris pH 8.0, 500 mM NaCl, 15 mM bME, 5 mM benzamidine, 0.01 mg / mL lysozyme, 0.05 mg / mL Dnase1, 5% (v / v) glycerol and Halt protease inhibitor tablets (Pierce)) using a Polytron PT2100 homogenizer. Homogenized cells were lysed with three passes using a Microfluidizer M-110Y (Mircofluidics). The lysate was clarified by centrifugation at 13,000 rpm in a JLA-16.250 rotor in a Beckman centrifuge. The clarified lysate was filtered through a 0.22 μm filter before being applied to a HisTrap Excel column (Cytiva). The resin was washed with 25 mM Tris pH 8.5, 0.5 M NaCl, 10 mM BME, 5% glycerol, 10 mM imidazole and MAGEB2 was eluted with 25 mM Tris pH 8.5, 0.5 M NaCl, 10 mM BME, 5% glycerol, 500 mM imidazole.

[0294] The captured affinity MAGEB2 was dialyzed against 20 mM Tris pH 8.5, 0.5 M NaCl, 5% (v / v) glycerol, 1 mM TCEP and diluted 25-fold into 20 mM Tris pH 8.5, 5% (v / v) glycerol, 1 mM TCEP before ion-exchange chromatography using Q-sepharose HP (Cytiva). Bound MAGEB2 was eluted from the column over 20 CV using a 20 mM to 500 mM NaCl gradient. Peak fractions were analyzed by SDS-PAGE and pooled based on the absence of a low molecular weight band. After addition of 1 mM EDTA and 0.1% CHAPS, the n-terminal 6xHis tag was removed from MAGEB2 using caspase 3 protease at a 1:100 (w / w) ratio. Digestion was allowed to proceed overnight at 4 °C. The digest was dialyzed against 20 mM Tris pH 8.5, 0.25 M NaCl, 1 mM TCEP to remove CHAPS and EDTA, followed by ion exchange chromatography.

[0295] Dialyzed MAGEB2 was diluted 10-fold in 20 mM Tris pH 8.5, 1 mM TCEP and applied to Q-sepharose HP resin (Cytiva). Bound MAGEB2 was eluted from the column over 20 CV using a 20 mM to 500 mM NaCl gradient. Peak fractions were analyzed by SDS-PAGE and pooled based on the absence of a high molecular weight band. This pool was biotinylated using recombinant BirA containing 50 mM bicine pH 8.3, 10 mM Mg acetate, 10 mM ATP and 50 μM d-biotin. The reaction was allowed to proceed overnight at 4°C and 5 μg of protein was analyzed by mass spectrometry (instrument and methods requested from Dylan) to monitor the addition of biotin to MAGEB2.

[0296] The biotinylation reaction was dialyzed against 20 mM Tris pH 8.5, 150 mM NaCl, 1 mM TCEP to remove excess ATP and biotin. After dialysis, the sample was diluted 10-fold with 20 mm Tris pH 8.5, 1 mM TCEP and applied to Q-sepharose HP (Cytiva). Bound MAGEB2 was eluted from the column over 20 CV using a 20 mM to 500 mM NaCl gradient. Peak fractions were analyzed by SDS-PAGE and pooled based on the presence of a 37 Kd band. Pooled fractions were concentrated using a 10 Kd MWCO spin filter (Sartorius). Concentrated MAGEB2 was dialyzed against 30 mM HEPES pH 8.2, 0.15 M NaCl, 1 mM TCEP, filtered through a 0.22 μm Posidyne syringe filter (Pall) and stored at -80°C.

[0297] During purification, the identity of intact, caspase-3 cleaved and biotinylated MAGEB2 was confirmed using mass spectrometry. Homogeneity of purified intermediates and final protein lots was measured by sizing HPLC analysis using a YARA 3 μm SEC 2000 column (Phenomenex) on an Agilent 1200 LC system. Protein concentration was determined by A280 absorbance using a Spectramax spectrophotometer (Molecular Devices).

[0298] Biotinylated MAGEB2 full-length protein was titrated 1:2 across six different defined antigen concentrations and then coated on six different LumAvidin beads in FACS buffer [1x PBS + 2% fetal bovine serum (Hyclone catalog number SH30396.03)] for 30 minutes at room temperature in the dark. The beads were then washed twice in FACS buffer by centrifugation at 3500 RPM for 2 minutes. The coated beads were resuspended, pooled together (6 portions) and added to rabbit B cell culture supernatants on Corning 3897 96-well V-bottom polystyrene plates at a final dilution of 1:2 and incubated overnight at room temperature in the dark. The next day, after washing twice in FACS buffer, Alexa 488-conjugated goat anti-rabbit IgG (Fc) (Jackson, catalog number 111-545-046) made in FACS buffer was added and incubated for 15 minutes at room temperature in the dark. After a final wash, plates were read on an iQue FACS instrument equipped with an Intellicyte autosampler to obtain flow cytometry measurements according to the manufacturer's recommendations, and data analysis was performed with Intellicyt ForeCyt® Enterprise Client Edition 6.2 (R3). The LA data was aligned with the sequence data to identify the 24 highest affinity, sequence diverse antibodies. A breakdown of the number of hits per region is shown in Table 12 below and in Figure 2 herein.

[0299] [Table 16]

[0300] Sequencing of antibody heavy and light chains Messenger RNA (mRNA) was purified from wells containing antibody-producing cells using the mRNA Catcher Plus kit (Invitrogen). The purified RNA was used to amplify the variable region (V) genes of the antibody heavy and light chains by reverse transcription-based cDNA synthesis followed by polymerase chain reaction (RT-PCR). Rabbit antibody gamma heavy chains were obtained using the Qiagen 1-step reverse transcriptase PCR kit (Qiagen). This method was used to generate first strand cDNA from an RNA template and then amplify the variable region of the gamma heavy chain by multiplex PCR. The 5' gamma chain specific primer was annealed to the signal sequence of the antibody heavy chain, while the 3' primer was annealed to a region of the gamma constant domain. Rabbit kappa light chains were obtained using the 1-step reverse transcriptase PCR kit (Qiagen). This method was used to generate first strand cDNA from an RNA template and then amplify the variable region of the kappa light chain by multiplex PCR. The 5' kappa light chain specific primer annealed to the signal sequence of the antibody light chain, while the 3' primer annealed to a region of the kappa constant domain.

[0301] The PCR products were sequenced and the amino acid sequences were bioinformatically deduced from the corresponding nucleic acid sequences. To ensure that any observed mutations were not the result of PCR, two additional independent RT-PCR amplification and sequencing cycles were performed for each sample. The resulting amino acid sequences were then analyzed to determine the germline sequence origin of the antibody and to identify any differences from the germline sequence. A comparison of each of the heavy and light chain sequences with their original germline sequences is shown in Sequence Table 4 herein. The amino acid sequences were used to group the clones by similarity.

[0302] The variable region kappa light chain DNA sequences of selected antibodies were generated as synthetic genes. These synthetic genes were cloned into a pTT5-based expression vector along with a rabbit kappa constant region. The variable region heavy chain DNA sequences of selected antibodies were generated as synthetic genes. These synthetic genes were cloned into a pTT5-based expression vector along with a rabbit IgG constant region. After cloning, these antibodies were expressed in PEI-transfected HEK2936E cells.

[0303] Example 3: Immunohistochemical Assay the purpose The specificity of 24 rabbit anti-human MAGEB2 antibodies for human MAGEB2 and potential cross-reactivity to other MAGE-B and MAGE-A family members is assessed by immunohistochemical (IHC) staining.

[0304] overview Twenty-four rabbit anti-human MAGEB2 supernatants were evaluated by IHC for binding to human MAGEB2 on transfected human MAGEB2+ control cells. After the initial screening assay, small batch purified antibody aliquots from four MAGEB2 clones (4G17, 1J15, 1C3, 1I14) with the desired attributes were evaluated by IHC on transfected human MAGE-A+ and MAGE-B+ family member control cells, normal human testis tissue, normal human tissue TMAs, and 13 human cancer cell lines with known endogenous MAGEB2 RNA expression levels. MAGEB2 antibody clones 1I14 and 1C3 displayed desirable properties for IHC.

[0305] Materials and Methods Control cells and tissues Negative control cells included parental Chinese Hamster Ovary (CHO) cells, CHO cells stably transfected with mCherry vector control (-Myc / DDK tag), and CHO cells stably transfected with mCherry-human DCAF4L2 (+Myc / DDK tag). Cross-reactive control cells included CHO cells transfected with mCherry vector control (+Myc / DDK tag) containing one of the human MAGE-B or MAGE-A family members (MAGEA1, MAGEA2, MAGEA3, MAGEA4, MAGEA5, MAGEA6, MAGEA8, MAGEA9, MAGEA11, MAGEA12, MAGEB1, MAGEB3, MAGEB4, MAGEB5, MAGEB6, MAGEB10, MAGEB16, MAGEB17, MAGEB18). Positive control cells included CHO cells transfected with mCherry vector containing human MAGE-B2 (+Myc / DDK tag) or CHO cells transfected with mCherry vector containing human MAGEB2 (open reading frame (ORF); -Myc / DDK tag) and 13 human cancer cell lines with known endogenous RNA expression levels of MAGEB2 (U266B1, T98G, KMM-1, U2OS, CFPAC-1, SCaBER, UACC-257, CFPAC-1, VMRC-LCD, NCI-H1703, UM-UC-3, NCI-H1395, NCIH82).

[0306] Cell pellets were fixed in 10% neutral buffered formalin and gel-coated with Histogel containing agarose / glycerin (catalog no. HG-4000-12, Thermo Fisher Scientific, Waltham, MA). TM The tissues were suspended in 5% CO and processed into paraffin blocks. Two FFPE normal human testis tissues and one FFPE normal human liver tissue obtained from the Human Tissue Science Center (HTSC) were used as positive and negative controls, respectively.

[0307] [Table 17]

[0308] [Table 18]

[0309] Tissue microarrays FFPE normal TMA slides (Normal Human Multiple Organ Tissue Microarray) were obtained from US BioMax (FDA999x, Crabbs Branch Way, Derwood, MD; https: / / www.biomax.US / FDA999x).

[0310] antibody Twenty-four human MAGEB2 clone supernatants (monoclonal rabbit IgG1 antibodies) were evaluated by IHC staining for binding to human MAGEB2 and potential cross-reactivity to other MAGE-A and MAGE-B family members on control cells. Four of the 24 human MAGEB2 clones (4G17, 1J15, 1C3, 1I14) were the most promising candidates for further evaluation of MAGEB2 specificity and sensitivity by IHC staining. Small batch purified antibody aliquots from the four MAGEB2 clones (4G17, 1J15, 1C3, 1I14) were evaluated by IHC on MAGE-A and MAGE-B family cross-reactive control cells, control testis tissue, and normal human tissue TMAs. Rabbit monoclonal IgG1 isotype control (EPR25A) was purchased from Abcam (catalogue number ab172730, lot number GR3235749-11, Cambridge, United Kingdom; https: / / www.abcam.com / rabbit-igg-monoclonal-epr25a-isotype-control-ab172730.html).

[0311] Immunohistochemistry assay FFPE samples were sectioned at 4 μm and mounted on positively charged glass slides. After mounting, paraffin sections were air-dried at room temperature. FFPE TMA samples were received as freshly sectioned and mounted on glass slides. Fully automated IHC assays were performed on a Discovery Ultra Stainer (Ventana Medical Systems, Tucson, AZ) using Ventana anti-rabbit HQ (catalog number 760-4815), Ventana anti-HQ HRP (catalog number 760-4820) and Ventana Antibody Diluent with Casein (catalog number 760-219) (Ventana Medical Systems, Tucson, AZ). Slides were baked in a Discovery Ultra Stainer at 60°C for 8 minutes, deparaffinized in Ventana Discovery washing solution (cat. no. 950-510) for 3 cycles (8 minutes each) at 69°C, and cell conditioning (target retrieval) was performed in CC1 buffer (cat. no. 950-500) at 95°C for 16 minutes (for cell arrays) or 32 minutes (tissues or TMAs). Slides were then incubated for 28 min with Background Sniper (catalog no. BS966M, Biocare Medical, Pacheco, CA) in a Ventana Option Prep kit (catalog no. 771-751), incubated for 12 min with CM inhibitor (part of the Ventana DAB kit (catalog no. 760-159)), incubated for 1 h at RT with MAGE-B2 antibody (working concentration of 0.1 μg / ml for cell pellets and 1-5 μg / ml for tissues) diluted in Ventana diluent with casein in a Ventana Prep kit (catalog no. 770-001), or incubated with the corresponding concentrations of isotype control rabbit IgG1 antibody. Slides were subsequently incubated with Ventana anti-rabbit HQ (cat. no. 760-4815) for 28 min at RT, Ventana anti-HQ HRP (cat. no. 760-4820) for 28 min at RT, and signal was detected with Ventana DAB chromogen kit (cat. no. 760-159) for 5 min at RT.Slides were counterstained with Ventana Hemoxilin II (Cat. No. 5266726001) for 8 minutes and Ventana Blue Reagent (Cat. No. 526676900) for 4 minutes. Slides were coverslipped and digitized using an Aperio AT2 scanner (Leica Biosystems Inc, Buffalo Grove, IL).

[0312] [Table 19]

[0313] result Twenty-four rabbit anti-human MAGEB2 antibody supernatants were evaluated by IHC for binding to MAGEB2 and potential cross-reactivity to other MAGE-A and MAGE-B family members. Five of the 24 human MAGEB2 antibody supernatants (5H20, 1J13, 1J14, 714, 7J6) did not detect MAGEB2 by IHC in human MAGEB2+ transfected cells. Nineteen of the 24 antibody supernatants detected MAGEB2 by IHC in human MAGEB2+ transfected cells and evaluated in other human MAGEA+ and MAGEB+ family member cross-reactive control cells. Four of the 19 supernatants (4G17, 1J15, 1C3, 1I14) showed the strongest IHC signal in human MAGEB2+ transfected control cells and the lowest / no cross-reactivity with other MAGE-A and MAGE-B family members and were selected as candidates for further evaluation.

[0314] Note that the 24 MAGEB2 antibody supernatants were not purified antibodies. The false positive cross-reactivity result of one of the discarded clones may be due to non-specific staining by the supernatant. Small batch purified antibody aliquots from four MAGEB2 clones (4G17, 1J15, 1C3, 1I14) were retested on transfected human MAGEA+ and MAGEB+ family member control cells and tested by IHC on control testicular tissue, normal tissue TMAs and 13 human cancer cell lines with known endogenous RNA expression levels of MAGEB2.

[0315] There was strong membrane / cytoplasmic staining in human MAGEB2+ cells transfected with four anti-MAGEB2 antibodies (4G17, 1J15, 1C3, 1I14) (Figure 3) and no cross-reactivity to other human MAGE-A+ and MAGE-B+ family member cross-reactive control cells (Figure 4, Table 15). In testis, strong MAGEB2 IHC staining was observed, characterized by cytoplasmic to membrane staining in spermatogonia. Nuclear staining in a subset of testicular germ cells was observed in 4G17, 1J15 and 1C3 clones, and immunostaining in Leydig cells was observed in 1J15 and 4G17 clones. Leydig staining is likely non-specific (Figure 5). Nuclear staining is likely specific, as it was only observed in testicular tissue, and nuclear localization may play a role in enhancing E2F transcriptional activity, cell proliferation and resistance to ribotoxic stress (Peche et al., 2015).

[0316] Four anti-MAGEB2 antibodies (4G17, 1J15, 1C3, 1I14) were evaluated in normal tissue TMAs. MAGEB2-specific immunostaining was observed only in testis cores. Immunostaining observed with 4G17, 1J15 and 1C3 clones in other tissues such as liver, adrenal gland and intestine was presumed to be non-specific. MAGEB2 antibody (1I14) showed the lowest non-specific / background immunostaining when compared to immunoglobulin G (IgG) isotype control (Tables 16, 17, 18, 19). Antibody clone 1I14 shows properties indicating it is an excellent IHC reagent for detecting MAGEB2 (Figure 6). Furthermore, down-titration of MAGEB2 (1C3) antibody completely reduced the non-specific background immunostaining observed in liver tissue, while MAGEB2-specific immunostaining in testis was still retained (Figure 7).

[0317] Anti-MAGEB2 antibodies 4G17, 1J15, 1C3, 1I14 detected various levels of MAGEB2 expression in 13 human cancer cell lines. MAGEB2 IHC expression frequency was high (~90%) in U266B1 cells, moderate (~70%) in T98G, KMM-1, U2OS cells, and undetectable in CFPAC-1, UACC-257, CFPAC-1, VMRC-LCD, NCI-H1703, UM-UC-3, NCI-H1395, NCI-H82 cell lines. MAGEB2 IHC expression frequency in SCaBER cells was inconsistent among the four MAGEB2 clones (4G17, 1J15, 1C3, 1i14), possibly due to different binding strengths of each antibody or non-optimized IHC assay conditions (Table 20).

[0318] conclusion Twenty-four rabbit anti-human MAGEB2 antibodies were evaluated for specificity to MAGEB2 and potential cross-reactivity to other MAGE family members by IHC staining on FFPE tissue. Nineteen of the 24 detected MAGEB2 by IHC in control MAGEB2+ cells. Four of the 19 antibodies (4G17, 1J15, 1C3, 1I14) were extensively tested and showed no cross-reactivity with other MAGE-A or MAGE-B family members, showed strong IHC signals in control testicular tissue, and detected MAGEB2 IHC signals in 13 human cancer cell lines with known endogenous RNA expression levels of MAGEB2. Three of the four MAGEB2 antibodies (4G17, 1J15, 1C3) showed some non-specific background IHC staining in liver and adrenal gland in normal human tissue TMAs. The data are summarized in Tables 15-20 herein.

[0319] array Human MAGEB2 amino acid sequence (SEQ ID NO:1): >sp|O15479|MAGB2_Human melanoma-associated antigen B2 OS=Homo sapiens OX=9606 GN=MAGEB2 PE=1 SV=3 [ka]

[0320] The complete amino acid sequences of the light and heavy chains of three exemplary monoclonal antibodies are as follows: >1C3_LC (sequence number 555) [ka] >1C3_HC (sequence number 556) [ka] >1I14_LC (sequence number 557) [ka] >1I14_HC (SEQ ID NO:558) [ka] >1H17_LC (SEQ ID NO:559) [ka] >1H17_HC (SEQ ID NO:560) [ka]

[0321] Further exemplary sequences of the present invention are provided in Tables 21-23 herein.

[0322] [Table 20]

[0323] [Table 21]

[0324] [Table 22]

[0325] [Table 23]

[0326] [Table 24]

[0327] [Table 25]

[0328] [Table 26]

[0329]

Table 27

[0330]

Table 28

[0331]

Table 29

[0332]

Table 30

[0333]

Table 31

[0334]

Table 32

[0335]

Table 33

[0336]

Table 34

[0337]

Table 35

[0338]

Table 36

[0339]

Table 37

[0340]

Table 38

[0341]

Table 39

[0342]

Table 40

[0343]

Table 41

[0344]

Table 42

[0345]

Table 43

[0346]

Table 44

[0347]

Table 45

[0348]

Table 46

[0349]

Table 47

[0350]

Table 48

[0351]

Table 49

[0352]

Table 50

[0353]

Table 51

[0354]

Table 52

[0355]

Table 53

[0356]

Table 54

[0357]

Table 55

[0358]

Table 56

[0359]

Table 57

[0360]

Table 58

[0361]

Table 59

[0362]

Table 60

[0363]

Table 61

[0364]

Table 62

[0365]

Table 63

[0366]

Table 64

[0367]

Table 65

[0368]

Table 66

[0369]

Table 67

[0370]

Table 68

[0371]

Table 69

[0372]

Table 70

[0373]

Table 71

[0374]

Table 72

Claims

1. An isolated antigen-binding construct that binds to MAGEB2, wherein the isolated antigen-binding construct binds to an epitope comprising a sequence selected from SEQ ID NOs: 2, 3, 4 or 388-554.

2. 2. The isolated antigen-binding construct of claim 1, comprising CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the sequence set forth in SEQ ID NO: 85, CDRL2 comprises the sequence set forth in SEQ ID NO: 86, CDRL3 comprises the sequence set forth in SEQ ID NO: 87, CDRH1 comprises the sequence set forth in SEQ ID NO: 229, CDRH2 comprises the sequence set forth in SEQ ID NO: 230, and CDRH3 comprises the sequence set forth in SEQ ID NO:

231.

3. 2. The isolated antigen-binding construct of claim 1, comprising CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the sequence set forth in SEQ ID NO: 73, CDRL2 comprises the sequence set forth in SEQ ID NO: 74, CDRL3 comprises the sequence set forth in SEQ ID NO: 75, CDRH1 comprises the sequence set forth in SEQ ID NO: 217, CDRH2 comprises the sequence set forth in SEQ ID NO: 218, and CDRH3 comprises the sequence set forth in SEQ ID NO:

219.

4. 2. The isolated antigen-binding construct of claim 1, comprising CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3, wherein CDRL1 comprises the sequence set forth in SEQ ID NO: 91, CDRL2 comprises the sequence set forth in SEQ ID NO: 92, CDRL3 comprises the sequence set forth in SEQ ID NO: 93, CDRH1 comprises the sequence set forth in SEQ ID NO: 235, CDRH2 comprises the sequence set forth in SEQ ID NO: 236, and CDRH3 comprises the sequence set forth in SEQ ID NO:

237.

5. 2. The isolated antigen-binding construct of claim 1, comprising a light chain variable region comprising the sequence set forth in SEQ ID NO:346 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:

347.

6. 2. The isolated antigen-binding construct of claim 1, comprising a light chain variable region comprising the sequence set forth in SEQ ID NO:338 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:

339.

7. 2. The isolated antigen-binding construct of claim 1, comprising a light chain variable region comprising the sequence set forth in SEQ ID NO:350 and a heavy chain variable region comprising the sequence set forth in SEQ ID NO:

351.

8. 2. The isolated antigen-binding construct of claim 1 which is a monoclonal antibody or an antibody fragment thereof.

9. 9. The isolated antibody of claim 8, which is of the IgG1 or IgG2 isotype.

10. 10. The isolated antigen-binding construct of any one of claims 1 to 9, which is bound to a labelling group.

11. A nucleic acid molecule encoding an antigen-binding construct or a fragment thereof according to any one of claims 1 to 9.

12. A vector comprising the nucleic acid molecule of claim 11.

13. A host cell comprising the nucleic acid molecule of claim 12.

14. 14. An antigen-binding construct or fragment thereof produced by the host cell of claim 13.

15. 10. A method of making an antigen-binding construct or fragment thereof according to any one of claims 1 to 9, comprising the step of preparing said antibody or fragment thereof from a host cell which secretes said antibody.

16. An isolated antibody or fragment thereof that competes with the antibody or fragment thereof of any one of claims 1 to 9 for binding to MAGEB2.

17. 1. A method for producing an antibody that binds to MAGEB2, comprising the steps of: Immunizing an animal with a peptide comprising a sequence selected from SEQ ID NO: 2, 3 or 4; isolating antibodies from said animal that bind to MAGEB2. The method includes:

18. 18. An antibody produced by the method of claim 17.

19. A composition comprising the antigen-binding construct of claim 1 for use in a method of treating a tumor in a subject, the method comprising the steps of determining that the subject will respond to treatment with an anti-MAGEB2 therapeutic agent by obtaining a sample from the subject comprising cells derived from the tumor, measuring the level of MAGEB2 in the sample using the antigen-binding construct of claim 1, and determining that the subject will respond to treatment with an anti-MAGEB2 therapeutic agent; administering to the subject an effective amount of the anti-MAGEB2 therapeutic agent; A composition comprising:

20. A composition for use in a method for identifying a subject as in need of an anti-MAGEB2 therapeutic, comprising the antigen-binding construct of claim 1, the method comprising: a) determining the level of MAGEB2 in a sample obtained from said subject using an antigen-binding construct according to claim 1; b) identifying the subject as being in need of the anti-MAGEB2 therapeutic if the level of MAGEB2 is increased compared to a control; A composition comprising:

21. A composition for use in a method for determining a treatment for a subject having a MAGEB2 positive tumor, comprising the antigen-binding construct of claim 1, the method comprising: Determining the level of MAGEB2 in a sample obtained from said subject using an antigen-binding construct according to claim 1; determining that the treatment comprises an anti-MAGEB2 therapeutic agent if the level of MAGEB2 is increased compared to a control; A composition comprising:

22. A composition for use in a method for determining the efficacy of treatment with an anti-MAGEB2 therapeutic in a subject, comprising the antigen-binding construct of claim 1, the method comprising: Determining the level of MAGEB2 in a sample obtained from the subject before treatment with an anti-MAGEB2 therapeutic agent and after treatment with an anti-MAGEB2 therapeutic agent using an antigen-binding construct according to claim 1; determining that the treatment is efficacious if the level of MAGEB2 positive tumor cells is reduced following treatment with the anti-MAGEB2 therapeutic agent; A composition comprising:

23. A composition for use in a method for diagnosing a subject having a tumor, comprising the antigen-binding construct of claim 1, the method comprising: a) determining the level of MAGEB2 in a sample obtained from said subject using an antigen-binding construct according to claim 1; b) diagnosing the subject as having a MAGEB2 positive tumor if the level of MAGEB2 is increased compared to a control; A composition comprising:

24. A composition for use in a method for identifying a subject having a MAGEB2 positive tumor, comprising the antigen-binding construct of claim 1, the method comprising: a) determining the level of MAGEB2 in a sample obtained from said subject using an antigen-binding construct according to claim 1; b) identifying the subject as having a MAGEB2 positive tumor if the level of MAGEB2 is increased compared to a control; A composition comprising:

25. A composition for use in a method for identifying a subject as in need of an anti-MAGEB2 therapeutic, comprising the antigen-binding construct of claim 1, the method comprising: a) determining the level of MAGEB2 in a sample obtained from said subject using an antigen-binding construct according to claim 1; b) identifying the subject as being in need of the anti-MAGEB2 therapeutic if the level of MAGEB2 is increased compared to a control; A composition comprising:

26. A composition for use in a method for determining a treatment for a subject having a MAGEB2 positive tumor, comprising the antigen-binding construct of claim 1, the method comprising: Determining the level of MAGEB2 in a sample obtained from said subject using an antigen-binding construct according to claim 1; determining that the treatment comprises an anti-MAGEB2 therapeutic agent if the level of MAGEB2 is increased compared to a control; A composition comprising:

27. The composition of any one of claims 19 to 26, wherein MAGEB2 is detected using an IHC assay.

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