ANTIBODY DRUG CONJUGATES BINDING TO HIGH-MANNOSE N-GLYCAN

FIELD OF THE INVENTION

The invention relates to a binding agent, a pharmaceutical composition, an isolated nucleic acid, a recombinant expression vector, a host cell, a method of treating cancer, autoimmune disease, inflammatory disorder or infection, a method of modulating growth of a cell population and a method for treating and/or modulating the growth and/or prophylaxis of tumor cells in humans or animals.

BACKGROUND OF THE INVENTION

Binding agents, such as antibodies and antibody-drug conjugates (antibodies conjugated to a drug payload molecule) provide targeted therapeutics with improved potential over traditional chemotherapy. Binding agents with improved specificity, internalization into target cells, cytotoxicity in the target cells and other beneficial properties are thus in demand.

SUMMARY

The binding agent according to the present invention is characterized by what is presented in claim 1.

The binding agent according to the present invention is characterized by what is presented in claim 2.

The pharmaceutical composition according to the present invention is characterized by what is presented in claim 35.

The isolated nucleic acid according to the present invention is characterized by what is presented in claim 36.

The recombinant expression vector according to the present invention is characterized by what is presented in claim 37.

The host cell according to the present invention is characterized by what is presented in claim 38.

The binding agent or the pharmaceutical composition for use as a medicament according to the present invention is characterized by what is presented in claim 39. The binding agent or the pharmaceutical composition for use in the treatment of cancer, autoimmune disease, inflammatory disorder or infection according to the present invention is characterized by what is presented in claim 40.

The method of treating cancer, autoimmune disease, inflammatory disorder or infection according to the present invention is characterized by what is presented in claim 41.

The method of modulating growth of a cell population according to the present invention is characterized by what is presented in claim 43.

The method for treating and/or modulating the growth and/or prophylaxis of tumor cells in humans or animals according to the present invention is characterized by what is presented in claim 45.

The binding agent according to the present invention is characterized by what is presented in claim 47.

The binding agent according to the present invention is characterized by what is presented in claim 48.

The pharmaceutical composition according to the present invention is characterized by what is presented in claim 50.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are included to provide a further understanding of the invention and constitute a part of this specification, illustrate embodiments of the invention and together with the description help to explain the principles of the invention. In the drawings:

Figure 1 shows MALDI-TOF MS image of N-glycans released from GCM014.

Figure 2 shows MALDI-TOF MS image of LC's of GCM014. Figure 3 shows SDS-PAGE of GCM023 ran in reduced (lane 1) and non-reduced (lane 2) conditions. The locations of the molecular weight markers are shown by the arrows.

Figure 4 shows MALDI-TOF MS image of N-glycans released from GCM023.

Figure 5 shows MALDI-TOF MS image of LC's of GCM023. Figure 6 shows MALDI-TOF MS image of double charged Fc's of GCM023. Figure 7 GCM005 binds to cell surface (HSC-2) at 4°C.

After incubation at 37°C, antibody is internalized and most of the staining is seen inside the cells while only very weak staining could be detected on the cell surface.

Figure 8 shows heavy and light chain amino acid sequences of the 2G12 antibody.

Figure 9 shows heavy and light chain amino acid sequences of the cetuximab and imgatuzumab antibodies.

Figure 10 shows heavy and light chain amino acid sequences of the matuzumab and nimotuzumab antibodies.

Figure 11 shows heavy and light chain amino acid sequences of the necitumumab and zalutumumab antibodies.

Figure 12 shows heavy and light chain amino acid sequences of the trastuzumab, margetuximab, and pertuzumab antibodies.

DETAILED DESCRIPTION OF THE INVENTION According to a first aspect of the invention there is provided a binding agent comprising a payload molecule and a first binding component comprising the complementarity determining regions of an antibody, wherein said antibody is capable of binding a high-mannose N-glycan.

According to a second aspect of the invention there is provided a binding agent comprising a first binding component comprising the complementarity determining regions of an antibody, wherein said antibody is capable of binding a high- mannose N-glycan; and a second binding component capable of binding an antigen other than the antigen which the first binding component is capable of binding.

High-mannose N-glycans typically comprise 5-9 or 6-9 mannose residues attached to two GlcNAc residues.

In an embodiment of the first or second aspect, said antibody is capable of binding an oligosaccharide structure comprising the structure according to formula I Formula !

wherein

(β-Ν-Asn) = β-Ν linkage to asparagine;

Rl = absent or Man 2 _n Man 6, wherein n = 0 or 1 ; R2 = absent or Man 2 _m Man 3, wherein m = 0 or 1 ; R3 = Man 2 _k Man 3, wherein k = 0, 1 or 2 ; with the proviso that at least two substituent groups selected from the group consisting of Rl, R2 and R3 comprise at least two Man residues.

In an embodiment, the high-mannose N-glycan comprises the structure according to formula I.

In this context, the term "binding agent" should be understood as any molecule that is capable of binding an antigen, i.e. specifically recognises an antigen. The antigen may be a high-mannose N-glycan, or it may be e.g. another antigen present on the surface of a tumor cell or a cancer cell. In an embodiment, the binding agent is capable of binding or capable of specifically binding a high-mannose N-glycan.

As known in the art (see e.g. "Essentials of Glycobiology", 2 ^nd edition, Ed. Varki, Cummings, Esko, Freeze, Stanley, Bertozzi, Hart & Etzler; Cold Spring Harbor Laboratory Press, 2009) and used herein, the term "glycan" should be understood to refer to homo- or heteropolymers of sugar residues, which may be linear or branched. In this context, "glycan" should also be understood to encompass a glycan component of a glycoconjugate, i.e. a molecule wherein a sugar moiety is covalently linked to at least one moiety, e.g. a glycoprotein, glycolipid, or proteoglycan. "N-glycan", a term also well known in the art, refers to a glycan conjugated by a β-Ν-linkage (nitrogen linkage through a β-glycosidic bond) to an asparagine (Asn) residue of a protein. N-glycans exist in various forms, but typically comprise two W-acetylglucosamine residues to which three or more mannose residues are attached.

Glycolipid and carbohydrate nomenclature is essentially according to recommendations by the IUPAC-IUB Commission on Biochemical Nomenclature (e.g. Carbohydrate Res. 1998, 312, 167; Carbohydrate Res. 1997, 297, 1; Eur. J. Biochem. 1998, 257, 293) .

The term "oligosaccharide structure" refers to such glycan structures or portions thereof, which comprise sugar residues. Such sugar residues may comprise e.g. mannose, N- acetylglucosamine, glucose, or fucose linked to each other through glycosidic bonds in a particular configuration.

In this context, the abbreviation "Man" should be understood as mannose; and "GlcNAc" refers to N- acetylglucosamine .

In this context, the term "cancer" should be understood as meaning "tumor" or "cancer cells". The term "tumor" is to be understood as meaning solid multicellular tumor tissues, including premalignant tissue, which is developing to a solid tumor and has cancer specific characteristics.

The notation of the oligosaccharide structure and the glycosidic bonds between the sugar residues comprised therein follows that commonly used in the art, e.g. "Man 2Man" should be understood as meaning two mannose residues linked by a covalent linkage between the first carbon atom of the first mannose residue to the second carbon atom of the second mannose residue linked by an oxygen atom in the alpha configuration. Furthermore, in this context, the notation of the oligosaccharide structure "Man8GlcNAc2" should be understood as meaning a structure comprising eight mannose residues linked to two W-acetylglucosamine residues.

The term "specifically binding" or any related expression as used herein refers to the ability of a binding agent, a first or second binding component or an antibody to discriminate between a specific antigen and any other antigen to the extent that, from a pool of a plurality of different antigens as potential binding partners, only the specific antigen is bound or significantly bound. As examples only, specific binding and/or kinetic measurements may be assayed by e.g. by utilizing surface plasmon resonance-based methods on a Biacore apparatus, by immunological methods such as ELISA or by e.g. protein or carbohydrate microarrays. The generation of a binding agent according to the first or second aspect may be done using standard techniques well known in the art. For instance, to select a binding agent or a binding component having a desired binding capability and specificity, phage display based on standard procedures may be used as disclosed in e.g. "Phage Display: A Laboratory Manual"; Ed. Barbas, Burton, Scott & Silverman; Cold Spring Harbor Laboratory Press, 2001. Standard procedures of recombinant antibody technologies may also be used to produce the binding agent.

In the context of the first aspect of the invention, the term "first binding component" should not be interpreted so that the binding agent would always comprise more than one binding component. In other words, in some embodiments according to the first aspect of the invention, the binding agent may comprise only the first binding component but no other binding components .

The binding agent may also comprise more than two binding components, for instance a third binding component.

A skilled person will understand that a binding component (first binding component, second binding component or both) may comprise or consist of one or more domains. A binding component may also comprise or consist of one or more polypeptides. For instance, a binding component may comprise a single domain, or it may be an antibody comprising one or more polypeptides, each of which in turn may comprise one or more domains .

The features of the embodiments described below may be combined with the first aspect of the invention as well as with the second aspect of the invention.

In an embodiment, the high-mannose N-glycan is selected from the group consisting of a Man6GlcNAc2 N-glycan, a Man7GlcNAc2 N-glycan, a Man8GlcNAc2 N-glycan, and a Man9GlcNAc2 N-glycan .

In an embodiment, the first binding component is capable of binding a high-mannose N-glycan.

In an embodiment, the antibody or the first binding component is capable of specifically binding a high-mannose N- glycan . In an embodiment, the antibody is PGT125, PGT126, PGT127, PGT128, PGT130 or TM10 or a modification or derivative thereof .

In the context of the first and second aspect, the antibodies PGT125, PGT126, PGT127, PGT128 and PGT130 should be understood as referring to the antibodies described in Walker et al. 2011, Nature 477:466-471.

In the context of the first and second aspect, the antibody TM10 should be understood as referring to the antibody described in Newsom-Davis et al . 2009, Cancer Res. 69:2018-25.

In an embodiment, the antibody is an antibody selected from the group consisting of PGT125, PGT126, PGT127, PGT128, PGT130 and TM10.

In an embodiment, the antibody is 2G12 or a modification or derivative thereof.

In the context of the first and second aspect, the term "2G12" is to be understood as meaning the domain exchanged human monoclonal IgGl antibody produced from the hybridoma cell line CL2 (described in e.g. US 5,911,989; Buchacher et al. 1994, AIDS Research and Human Retroviruses, 10(4), 359-369; and Trkola et al. 1996, Journal of Virology, 70(2), 1100-1108), and any synthetically, e.g. recombinantly, produced antibody having an identical sequence of amino acids, including any antibody fragment thereof having at least the antigen-binding portions of the heavy and light chain variable region domains to the full- length antibody, such as the 2G12 domain exchanged Fab fragment (described in e.g. US 20050003347; Calarese et al. 2003, Science, 300, 2065-2071), or a modification or derivative thereof .

In an embodiment, the first binding component comprises the heavy chain complementarity determining regions 1-3 having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as compared to the heavy chain complementarity determining regions 1-3 of PGT125, PGT126, PGT127, PGT128, PGT130 or TM10, and the light chain complementarity determining regions 1-3 of PGT125, PGT126, PGT127, PGT128, PGT130 or TM10 or light chain complementarity determining regions 1-3 having at least about 80 90 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity as compared to light chain complementarity determining regions 1-3 of PGT125, PGT126, PGT127, PGT128, PGT130 or TM10.

In an embodiment, the first binding component comprises heavy chain complementarity determining regions 1-3 and/or light chain complementarity determining regions having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NOs: 1 - 4, amino acid sequence KAS and SEQ ID NO: 6.

In an embodiment, the first binding component comprises the heavy chain complementarity determining regions HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3) and the light chain complementarity determining regions LCDR1 (SEQ ID NO: 4), LCDR2 (amino acid sequence KAS) and LCDR3 (SEQ ID NO: 6) of the antibody 2G12.

LCDR2 of the antibody 2G12 has the amino acid sequence

KAS (i.e. Lys Ala Ser) .

In an embodiment, the first binding component is a scFv, a single domain antibody, an Fv, a VHH antibody, a diabody, a tandem diabody, a Fab, a Fab', or a F(ab)2.

The first binding component may be e.g. an scFv, a single domain antibody, an Fv, a VHH antibody, a diabody, a tandem diabody, a Fab, a Fab', a F(ab')2, a Db, a dAb-Fc, a taFv, a scDb, a dAb ₂ , a DVD-Ig, a Bs (scFv) ₄ -IgG, a taFv-Fc, a scFv-Fc- scFv, a Db-Fc, a scDb-Fc, a scDb-C _H 3, or a dAb-Fc-dAb.

The second binding component may be e.g. an scFv, a single domain antibody, an Fv, a VHH antibody, a diabody, a tandem diabody, a Fab, a Fab', a F(ab')2 _/ a Db, a dAb-Fc, a taFv, a scDb, a dAb ₂ , a DVD-Ig, a Bs (scFv) ₄ -IgG, a taFv-Fc, a scFv-Fc- scFv, a Db-Fc, a scDb-Fc, a scDb-C _H 3, or a dAb-Fc-dAb.

The binding agent may be present in monovalent monospecific, multivalent monospecific, bivalent monospecific, or multivalent multispecific forms.

In an embodiment, the first binding component is a human antibody or a humanized antibody. In an embodiment, the second binding component is a human antibody or a humanized antibody. In this context, the term "human antibody", as it is commonly used in the art, is to be understood as meaning antibodies having variable regions in which both the framework and complementarity determining regions (CDRs) are derived from sequences of human origin. In this context, the term

"humanized antibody", as it is commonly used in the art, is to be understood as meaning antibodies wherein residues from a CDR of an antibody of human origin are replaced by residues from a CDR of a nonhuman species (such as mouse, rat or rabbit) having the desired specificity, affinity and capacity.

In an embodiment, the first binding component is a scFv comprising the sequence set forth in SEQ ID NO: 7 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 7.

In an embodiment, the first binding component is a scFv comprising the sequence set forth in SEQ ID NO: 8 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 8.

In an embodiment, the first binding component is a scFv comprising the sequence set forth in SEQ ID NO: 9 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 9.

In an embodiment, the first binding component is a scFv comprising the sequence set forth in SEQ ID NO: 10 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 10.

In an embodiment, the scFv comprising the sequence set forth in SEQ ID NO: 7, 8, 9 or 10, or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to the SEQ ID NO: 7, 8, 9 or 10 is capable of binding a high-mannose N-glycan.

In an embodiment, the first binding component comprises a heavy chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 11 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 11.

In an embodiment, the heavy chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 11 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 11 is capable of binding a high-mannose N-glycan.

In an embodiment, the first binding component comprises a light chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 12 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 12.

In an embodiment, the light chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO:

12 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 12 is capable of binding a high-mannose N-glycan .

In an embodiment, the first binding component comprises a heavy chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 11 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 11 and light chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 12 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to the SEQ ID NO: 12, and optionally the light chain constant domain of the antibody 2G12 and/or one or more heavy chain constant domains of the antibody 2G12 selected from the group consisting of CHI, CH2 and CH3 of the antibody 2G12.

In an embodiment, the heavy chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 11 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 11 and/or the light chain variable region of the antibody 2G12 comprising a sequence set forth in SEQ ID NO: 12 or a sequence having at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity as compared to SEQ ID NO: 12 is capable of binding a high-mannose N-glycan. In an embodiment, the first binding component or the antibody 2G12 comprises a heavy chain variable region comprising a sequence set forth in SEQ ID NO: 11 and a light chain variable region comprising a sequence set forth in SEQ ID NO: 12.

In an embodiment, the first binding component comprises the light chain constant domain of the antibody 2G12 and/or one or more heavy chain constant domains of the antibody 2G12 selected from the group consisting of CHI, CH2 and CH3 of the antibody 2G12.

In an embodiment, the first binding component comprises one or more regions or domains selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of a sequence corresponding to the light chain of an Ig molecule; and/or one or more regions or domains selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of a sequence corresponding to the heavy chain of an Ig molecule; wherein the first binding component comprises one or more introduced N- glycosylation sites in any region or domain selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of the light chain; and/or one or more introduced N-glycosylation sites in any region or domain in any region or domain selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of the heavy chain.

In an embodiment, the first binding component comprises the antibody 2G12.

In an embodiment, the first binding component is or consists of the antibody 2G12.

In an embodiment, one or more amino acid residues of the first binding component are substituted by substitutions selected from the group consisting of I19N, E89N, G165S, Q181N, L188N, S196N, and L199N as compared to SEQ ID NO: 13.

In an embodiment, the first binding component comprises the 2G12 heavy chain having a sequence set forth in SEQ ID NO: 13, wherein one or more amino acid residues are substituted by substitutions selected from the group consisting of I19N, F78S, E89N, G165S, Q181N, L188N, S196N, and L199N as compared to SEQ ID NO: 13.

The substitution I19N should be understood as referring to a substitution of the amino acid residue I at the position 19 of the sequence set forth in SEQ ID NO: 13 to the amino acid residue N. Other substitutions described in this specification are indicated in the same manner. If the first binding component comprises a portion but not the full sequence of e.g. the heavy chain set forth in SEQ ID NO: 13, a skilled person is capable of determining a corresponding amino acid residue in the first binding component and making a corresponding substitution.

One or more of the substitutions may provide improved properties to the binding agent. For instance, the substitution I19N and/or F78S may improve the solubility of the binding agent, render the production of the binding agent easier and/or improve the yield of the binding agent. One or more of the substitutions may introduce an additional N-glycosylation site. The additional N-glycosylation sites may be utilized by incorporating therein e.g. N-glycans having beneficial properties or additional payload molecules.

In an embodiment, one or more amino acid residues of the first binding component are substituted by substitutions selected from the group consisting of T18N, L154S, Q160N S174N and T180N as compared to SEQ ID NO: 14.

In an embodiment, the first binding component comprises the 2G12 light chain having a sequence set forth in in SEQ ID NO: 14, wherein one or more amino acid residues are substituted by substitutions selected from the group consisting of T18N, L154S, Q160N S174N and T180N as compared to SEQ ID NO: 14.

In an embodiment, the first binding component comprises the sequences set forth in SEQ ID NOs : 15 and SEQ ID NO: 12.

In an embodiment, the first binding component comprises the sequences set forth in SEQ ID NOs: 15 and SEQ ID NO: 14.

In an embodiment, the first binding component comprises the sequences set forth in SEQ ID NOs: 16 and SEQ ID NO: 12.

In an embodiment, the first binding component comprises the sequences set forth in SEQ ID NOs: 16 and SEQ ID NO: 14.

In an embodiment, the first binding component comprises the sequences set forth in SEQ ID NOs: 17 and SEQ ID NO: 12. In an embodiment, the first binding component comprises the sequences set forth in SEQ ID NOs : 17 and SEQ ID NO: 14.

In an embodiment, the binding agent comprises a detection-enabling molecule. Examples of detection-enabling molecules are molecules conveying affinity such as biotin or a

His tag comprising at least five histidine (His) residues; molecules that have enzymatic activity such as horseradish peroxidase (HRP) or alkaline phosphatase (AP) ; various fluorescent molecules such as FITC, TRITC, and the Alexa and Cy dyes; gold; radioactive atoms or molecules comprising such; chemiluminescent or chromogenic molecules and the like, which molecules provide a signal for visualization or quantitation.

The detection-enabling molecule may be covalently bound to the binding agent, to the first binding component and/or to the second binding component, or it may be bound indirectly thereto e.g. via a suitable linker group. The linker group may be e.g. any linker described in this specification.

In an embodiment according to the first aspect, the binding agent comprises a second binding component capable of binding an antigen other than the antigen which the first binding component is capable of binding.

In the context of both the first aspect and the second aspect of the invention, the second binding component is capable of binding an antigen that is different or other than the antigen which the first binding component is capable of binding.

The second binding component may thus have a different binding specificity than the first binding component. The antigen that the second binding component is capable of binding may be e.g. an antigen present on the surface of a tumor cell or cancer cell .

In an embodiment, the second binding component is capable of binding an antigen other than a high-mannose N- glycan .

In an embodiment, the second binding component is capable of binding a cell surface antigen.

In an embodiment, the cell surface antigen is a tumor antigen and/or a cancer antigen. In an embodiment, the tumor antigen/a cancer antigen may include, but is not limited to, CD2, CD3, CD4, CD5, CD6, CD11, CD8, CDlla, CD19, CD20, CD22, CD25, CD26, CD30, CD33, CD34, CD37, CD38, CD40, CD44, CD46, CD52, CD56, CD79, CD105, and CD138; members of the ErbB receptor family, such as epidermal growth factor receptor 1 (EGFR) , epidermal growth factor receptor 2 (HER2/neu), HER3 or HER4 receptor; cell adhesion molecules, such as LFA-1, Macl, pl50.95, VLA-4, ICAM-1, VCAM, EpCAM, alpha4/beta7 integrin, and alpha v/beta3 integrin including either alpha or beta subunits thereof; growth factors, such as VEGF; tissue factor (TF) ; tumor necrosis factor alpha (TNF- ) ; human vascular endothelial growth factor (VEGF) ; glycoprotein Ilb/IIIa; TGF-beta; alpha interferon (alpha-IFN) ; an interleukin, such as IL-8; an interleukin receptor, such as IL-2 receptor; cancer-associated high-mannose type N-glycans; blood group antigens Apo2, death receptor; flk2/flt3 receptor; CTLA-4; transferrin receptor; or a cancer-associated glycan structure, such as Lewis y or GD3.

The features of the embodiments described below may be combined with the first aspect of the invention as well as with the second aspect of the invention.

The binding agent according to the first or second aspect may be a bispecific binding agent, such as a bispecific antibody molecule. A bispecific binding agent contains both the first binding component and the second binding component and is thus capable of binding to two different antigens or epitopes. The binding agent may also be a multispecific binding agent, such as a multispecific antibody molecule.

A skilled person is well aware that there are a number of ways to create a bispecific or a multispecific binding agent e.g. by recombinant technology. Various formats of bispecific binding agents, such as bispecific antibodies, are described e.g. in Kontermann RE (2012) Dual targeting strategies with bispecific antibodies, mAbs 4:182-197. The bispecific binding agent or a protein moiety thereof may comprise e.g. a bispecific IgG ₂ , IgG, IgG, IgGscFv, scFv-IgG, F(ab')2, F (ab' ) 2-scFv, diabody, dual-affinity re-targeted antibody (DART) , tandem scFv, Fab-scFv, taFv, scDb, dAb ₂ , DVD-Ig, Bs (scFv) ₄ -IgG, taFv-Fc, scFv-Fc-scFv, db-Fc, scDb-Fc, scDb-C _H 3, or dAb-Fc-dAb. In an embodiment, the binding agent comprises IgG ₂ . In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the IgG ₂ . In an embodiment, additional N-glycosylation sites are introduced into the light chain of the IgG ₂ . In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the IgG ₂ . In an embodiment, additional N-glycosylation sites are introduced into the light chain of the IgG ₂ . In an embodiment, additional N- glycosylation sites are introduced into the heavy chains and light chains of the IgG ₂ .

In an embodiment, the binding agent comprises a bispecific IgG. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the bispecific IgG. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the bispecific IgG. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the bispecific IgG. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the bispecific IgG. In an embodiment, additional N- glycosylation sites are introduced into the heavy chains and light chains of the bispecific IgG.

In an embodiment, the binding agent comprises an IgG- scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the IgG-scFv. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the IgG-scFv. In an embodiment, additional N- glycosylation sites are introduced into the heavy chain of the IgG-scFv. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the IgG-scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chains and light chains of the IgG-scFv.

In an embodiment, the binding agent comprises an scFv- IgG. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the scFv-IgG. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the scFv-IgG. In an embodiment, additional N- glycosylation sites are introduced into the heavy chain of the scFv-IgG. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the scFv-IgG. In an embodiment, additional N- glycosylation sites are introduced into the heavy chains and light chains of the scFv- IgG.

In an embodiment, the binding agent comprises a bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the light chain of the bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the light chain of the bispecific F(ab' )2- In an embodiment, additional N- glycosylation sites are introduced into the heavy chains and light chains of the bispecific F(ab' )2- In an embodiment, the binding agent comprises an

F (ab' ) 2~scFv . In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the light chain of the bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the bispecific F(ab' )2- In an embodiment, additional N- glycosylation sites are introduced into the light chain of the bispecific F(ab' )2- In an embodiment, additional N-glycosylation sites are introduced into the heavy chains and light chains of the bispecific F(ab' )2-

In an embodiment, the binding agent comprises a diabody. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the diabody. In an embodiment, additional N-glycosylation sites are introduced into light chain of the diabody. In an embodiment, additional N- glycosylation sites are introduced into the heavy chain of the diabody. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the diabody. In an embodiment, additional N-glycosylation sites are introduced into the heavy chains and light chains of the diabody.

In an embodiment, the binding agent comprises a dual- affinity re-targeted antibody (DART) . In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the DART. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the DART. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the DART. In an embodiment, additional N- glycosylation sites are introduced into the light chain of the DART. In an embodiment, additional N-glycosylation sites are introduced into the heavy chains and light chains of the DART.

In an embodiment, the binding agent comprises a tandem scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the tandem scFv. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the tandem scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the tandem scFv. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the tandem scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chains and light chains of the tandem scFv.

In an embodiment, the binding agent comprises a Fab- scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chain of the Fab-scFv. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the Fab-scFv. In an embodiment, additional N- glycosylation sites are introduced into the heavy chain of the Fab-scFv. In an embodiment, additional N-glycosylation sites are introduced into the light chain of the Fab-scFv. In an embodiment, additional N-glycosylation sites are introduced into the heavy chains and light chains of the Fab-scFv.

The first binding component and the second binding component may be functionally linked e.g. by chemical coupling, fusion, or non-covalent association.

In an embodiment, the first binding component and the second binding component are fused. The binding agent thus comprises a fusion polypeptide, wherein the fusion polypeptide comprises both the first binding component and the second binding component. A skilled person is well aware of a number of methods of producing fusion polypeptides. The C-terminus of the first binding component may be fused to the N-terminus of the second binding component, or the C-terminus of the second binding component may be fused to the N-terminus of the first binding component. The first binding component and the second binding component may be fused directly or indirectly, e.g. via a linker sequence.

In an embodiment, the first binding component and the second binding component are chemically cross-linked. They may be chemically crosslinked e.g. via a suitable linker group. The length of the linker group may be selected based on the first and second binding component to be joined. Suitable linker groups may be, for example, m-maleimidobenzoyl-N- hydroxysuccinimide ester or disuccinimidyl suberate.

In an embodiment, the second binding component is capable of binding human EGFRl .

In an embodiment, the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR, and LCDR3 of an anti-EGFRl antibody .

In an embodiment, the first binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 of 2G12 and the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 of 2G12, and the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 of an anti-EGFRl antibody.

In an embodiment, the second binding component is an anti-EGFRl antibody or an EGFRl binding fragment thereof.

In an embodiment, the second binding component comprises a heavy chain variable region and a light chain variable region of an anti-EGFRl antibody.

In an embodiment, the anti-EGFRl antibody is selected from the group consisting of cetuximab, imgatuzumab, matuzumab, nimotuzumab, necitumumab, panitumumab, and zalutumumab.

In an embodiment, the anti-EGFRl antibody is cetuximab.

In an embodiment, cetuximab comprises the sequences set forth in SEQ ID NO:s 18 and 19.

In one embodiment, additional N-glycosylation sites are introduced into the cetuximab heavy chain. In one embodiment, the cetuximab heavy chain comprises one or more substitutions selected from the group consisting of G161S, Q177N, L184N, S192N, and L195N as compared to SEQ ID NO: 18.

In one embodiment, additional N-glycosylation sites are introduced into the cetuximab light chain. In one embodiment, cetuximab light chain comprises one or more substitutions selected from the group consisting of R18N, L154S, Q160N, S174N, and T180N as compared to SEQ ID NO: 19.

In an embodiment, the anti-EGFRl antibody is imgatuzumab .

In an embodiment, imgatuzumab comprises the sequences set forth in SEQ ID NO:s 20 and 21.

In an embodiment, additional N-glycosylation sites are introduced into the imgatuzumab heavy chain.

In an embodiment, the imgatuzumab heavy chain comprises one or more substitutions selected from the group consisting of E80N, G164S, Q178N, L185N, S193N, and L196N as compared to SEQ ID NO: 20.

In an embodiment, additional N-glycosylation sites are introduced into imgatuzumab light chain.

In an embodiment, imgatuzumab light chain comprises one or more substitutions selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 21.

In an embodiment, the anti-EGFRl antibody is matuzumab. In an embodiment, matuzumab comprises the sequences set forth in SEQ ID NO:s 22 and 23.

In an embodiment, additional N-glycosylation sites are introduced into the matuzumab heavy chain.

In an embodiment, the matuzumab heavy chain comprises one or more substitutions selected from the group consisting of E89N, G165S, Q179N, L186N, S194N, and L197N as compared to SEQ ID NO: 22.

In an embodiment, additional N-glycosylation sites are introduced into the matuzumab light chain.

In an embodiment, the matuzumab light chain comprises one or more substitutions selected from the group consisting of R18N, L159S, Q165N, S179N and T185N as compared to SEQ ID NO: 23. In an embodiment, nimotuzumab comprises the sequences set forth in SEQ ID NO:s 24 and 25.

In an embodiment, additional N-glycosylation sites are introduced into the nimotuzumab heavy chain. In an embodiment, the nimotuzumab heavy chain comprises one or more substitutions selected from the group consisting of E74N, E89N, G165S, Q181N, L188N, S196N, and L199N as compared to SEQ ID NO: 24.

In an embodiment, additional N-glycosylation sites are introduced into the nimotuzumab light chain.

In one embodiment, the nimotuzumab light chain comprises one or more substitutions selected from the group of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 25.

In an embodiment, the anti-EGFRl antibody is necitumumab. In an embodiment, necitumumab comprises the sequences set forth in SEQ ID NO:s 26 and 27.

In an embodiment, additional N-glycosylation sites are introduced into the necitumumab heavy chain.

In an embodiment, the necitumumab heavy chain comprises one or more substitutions selected from the group consisting of A89N, G165S, Q179N, L186N, S194N and L197N as compared to SEQ ID NO: 26.

In an embodiment, additional N-glycosylation sites are introduced into the necitumumab light chain.

In an embodiment, the necitumumab light chain comprises one or more substitutions selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 27.

In an embodiment, the antibody is panitumumab. In one embodiment, additional N-glycosylation sites can be introduced into heavy and/or light chains of panitumumab, and any other antibody herein, as described in, for example, W097/34632 and/or W095/15769. As described in W097/34632, additional N-glycosylation sites may be those of depicted in Fig 12 and corresponding to HCN1, HCN2, HCN3, HCN4, and/or HCN5 for heavy chain, and KCN1, KCN2, KCN3, and/or KCN4 for kappa light chain. An additional N-glycosylation site in an antibody may mean one or more non-Asn297 N-glycosylation sites. The non- Asn297 N-glycosylation sites can be present in or be introduced into a heavy and/or a light chain.

In an embodiment, the anti-EGFRl antibody is zalutumumab .

In an embodiment, zalutumumab comprises the sequences set forth in SEQ ID NO:s 28 and 29.

In an embodiment, additional N-glycosylation sites are introduced into the zalutumumab heavy chain.

In an embodiment, the zalutumumab heavy chain comprises one or more substitutions selected from the group consisting of E89N, G169S, Q183N, L190N, S198N, and L201N as compared to SEQ ID NO: 28.

In an embodiment, additional N-glycosylation sites are introduced into the zalutumumab light chain.

In an embodiment, the zalutumumab light chain comprises one or more substitutions selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 29.

In an embodiment, the anti-EGFRl antibody or its EGFR1 binding fragment comprises one or more regions or domains selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of a sequence corresponding to the light chain of an anti-EGFRl Ig molecule; and/or one or more regions or domains selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of sequence corresponding to the heavy chain of an anti-EGFRl Ig molecule; wherein the anti-EGFRl antibody or its EGFR1 binding fragment comprises one or more introduced N-glycosylation sites in any region or domain selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of the light chain; and/or one or more introduced N-glycosylation sites in any region or domain in any region or domain selected from the group consisting of the complementarity determining regions, framework regions, variable region, and constant domains of the heavy chain.

In an embodiment, the first binding component is a scFv derived from 2G12, and the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of an anti-EGFRl antibody.

In an embodiment, the scFv derived from 2G12 is a scFv comprising the heavy chain complementarity determining regions HCDR1 (SEQ ID NO: 1), HCDR2 (SEQ ID NO: 2) and HCDR3 (SEQ ID NO: 3) and the light chain complementarity determining regions LCDR1 (SEQ ID NO: 4), LCDR2 (amino acid sequence KAS) and LCDR3 (SEQ ID NO: 6) of 2G12.

In an embodiment, the scFv derived from 2G12 comprises the sequence set forth in SEQ ID NO: 7.

In an embodiment, the scFv derived from 2G12 comprises a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs : 7 - 10.

In an embodiment, the first binding component is a scFv comprising the sequence set forth in SEQ ID NO: 7, and the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of an anti-EGFRl antibody.

In an embodiment, the first binding component is a scFv comprising a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 7 - 10, and the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR2 and LCDR3 of an anti-EGFRl antibody.

In an embodiment, the scFv derived from 2G12 is fused to the C-terminus of an anti-EGFRl antibody or an EGFRl binding fragment thereof.

In an embodiment, the scFv derived from 2G12 is fused to the C-terminus of the heavy chain of an anti-EGFRl antibody or an EGFRl binding fragment thereof.

In an embodiment, the scFv derived from 2G12 is fused to the N-terminus of the heavy chain of an anti-EGFRl antibody or an EGFRl binding fragment thereof.

In an embodiment, the scFv derived from 2G12 is fused to the N-terminus of an anti-EGFRl antibody or an EGFRl binding fragment thereof. In an embodiment, the second binding component comprises at least one of the following:

(i) the cetuximab heavy chain having a sequence set forth in SEQ ID NO: 18, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of G161S, Q177N, L184N, S192N and L195N as compared to SEQ ID NO: 18;

(ii) imgatuzumab heavy chain having a sequence set forth in SEQ ID NO: 20, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of E80N, G164S, Q178N, L185N, S193N, and L196N as compared to SEQ ID NO: 20;

(iii) matuzumab heavy chain having a sequence set forth in SEQ ID NO: 22, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of E89N, G165S, Q179N, L186N, S194N, and L197N as compared to SEQ ID NO: 22;

(iv) nimotuzumab heavy chain having a sequence set forth in SEQ ID NO: 24, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of E74N, E89N, G165S, Q181N, L188N, S196N, and L199N as compared to SEQ ID NO: 24;

(v) necitumumab heavy chain having a sequence set forth in SEQ ID NO: 26, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of A89N, G165S, Q179N, L186N, S194N, and L197N as compared to SEQ ID NO: 26;

(vi) panitumumab heavy chain, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of E to N substitution at about amino acid 91, G to S substitution at about amino acid 161 (in sequence NSG) , Q to N at about amino acid 177 (in sequence QSS), L to N at about amino acid 184 (in sequence LSS), S to N at about amino acid 192 (in sequence SSS) , and L to N at about amino acid 195 (in sequence LGT) as compared to the panitumumab heavy chain sequence; or

(vii) zalutumumab heavy chain having a sequence set forth in in SEQ ID NO: 28, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of E89N, G169S, Q183N, L190N, S198N, and L201N as compared to SEQ ID NO: 28.

In an embodiment, the second binding component comprises at least one of the following:

(i) the cetuximab light chain having a sequence set forth in in SEQ ID NO: 19, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of R18N, L154S, Q160N, S174N, and T180N as compared to SEQ ID NO: 19;

(ii) the imgatuzumab light chain having a sequence set forth in in SEQ ID NO: 21, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 21;

(iii) the matuzumab light chain having a sequence set forth in in SEQ ID NO: 23, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 23;

(iv) the nimotuzumab light chain having a sequence set forth in in SEQ ID NO: 25, comprises one or more substitutions selected from the group of: R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 25;

(v) the necitumumab light chain having a sequence set forth in in SEQ ID NO: 27, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 27;

(vi) the panitumumab light chain wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of R to N substitution at about amino acid 18, L to S substitution at about amino acid 154 (in sequence NAL) , Q to N substitution at about amino acid 160 (in sequence QES) , S to N substitution at about amino acid 174 (sequence SLS -> NLS) , and T to N substitution at about amino acid 180 (in sequence TLS) as compared to the panitumumab light chain sequence; or

(vii) the zalutumumab light chain having a sequence set forth in in SEQ ID NO: 29, wherein one or more amino acid residues are substituted by a substitution selected from the group consisting of R18N, L159S, Q165N, S179N, and T185N as compared to SEQ ID NO: 29.

In an embodiment, the second binding component is capable of binding human ERBB2/HER2.

In an embodiment, the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2 and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR, and LCDR3 of an anti-ERBB2 /HER2 antibody.

In an embodiment, the first binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 of 2G12 and the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 of 2G12, and the second binding component comprises the heavy chain complementarity determining regions HCDR1, HCDR2, and HCDR3 and the light chain complementarity determining regions LCDR1, LCDR2, and LCDR3 of an anti-ERBB2 /HER2 antibody.

In an embodiment, the second binding component is an anti- ERBB2/HER2 antibody or an ERBB2/HER2 binding fragment thereof .

In an embodiment, the second binding component comprises a heavy chain variable region and a light chain variable region of an anti-ERBB2 /HER2 antibody.

In an embodiment, the anti-EGFRl antibody is selected from the group consisting of trastuzumab, ertumaxomab, margetuximab, and pertuzumab.

In an embodiment, the anti-ERBB2 /HER2 antibody is trastuzumab. In an embodiment, trastuzumab comprises the sequences set forth in SEQ ID NO:s 30 and 31. In an embodiment, additional N-glycosylation sites are introduced into the trastuzumab heavy chain. In an embodiment, additional N- glycosylation sites are introduced into the trastuzumab light chain .

In an embodiment, the anti-ERBB2 /HER2 antibody is ertumaxomab. In an embodiment, additional N-glycosylation sites are introduced into the ertumaxomab heavy chain. In an embodiment, additional N-glycosylation sites are introduced into the ertumaxomab light chain. In an embodiment, the anti-ERBB2 /HER2 antibody is margetuximab . In an embodiment, margetuximab comprises the sequences set forth in SEQ ID NO:s 32 and 33. In an embodiment, additional N-glycosylation sites are introduced into the margetuximab heavy chain. In an embodiment, additional N- glycosylation sites are introduced into the margetuximab light chain .

In an embodiment, the anti-ERBB2 /HER2 antibody is pertuzumab. In an embodiment, pertuzumab comprises the sequences set forth in SEQ ID NO:s 34 and 35. In an embodiment, additional N-glycosylation sites are introduced into the pertuzumab heavy chain. In an embodiment, additional N-glycosylation sites are introduced into the pertuzumab light chain.

In an embodiment, the first binding component and/or the second binding component comprises one or more additional stabilizing amino acid substitutions.

In an embodiment, the first binding component and/or the second binding component comprises one or more additional stabilizing amino acid substitutions in the heavy chain.

In an embodiment, the binding agent comprises a fusion polypeptide comprising one or more additional stabilizing amino acid substitutions.

In an embodiment, the first binding component and/or the second binding component comprises one or more additional stabilizing amino acid substitutions, such as an additional cysteine residues.

In an embodiment, the first binding component and/or the second binding component comprises one or more additional stabilizing amino acid substitutions, such as an additional cysteine residues in the scFv.

Exemplary stabilizing amino acid substitutions include, but are not limited to, an additional cysteine residue in the heavy chain variable and/or light chain variable domain of an scFv. Such an additional cysteine residue may be capable of forming a sulphur bridge with another cysteine residue, for instance another additional cysteine residue in the scFv, and thus stabilize the structure.

In an embodiment, a stabilizing amino acid substitution corresponds to G44C in a heavy chain of an Ig molecule. In an embodiment, a stabilizing amino acid substitution corresponds to G100C in a light chain of an Ig molecule .

In an embodiment, stabilizing amino acid substitutions correspond to G44C in a heavy chain and G100C in a light chain of an Ig molecule. In an embodiment, a stabilizing amino acid substitution is G44C in a heavy chain of 2G12 (scFv) and G100C in a light chain of 2G12 (scFv) .

In an embodiment, the binding agent comprises a fusion polypeptide comprising the nimotuzumab heavy chain fused to a scFv derived from 2G12. In this context, the scFv derived from 2G12 may be any scFv derived from 2G12 described in this specification .

In an embodiment, the binding agent comprises a fusion polypeptide having a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs : 36, 37 and 38.

In an embodiment, the binding agent comprises a fusion polypeptide comprising a sequence selected from the group consisting of the sequences set forth in SEQ ID NOs: 36, 37 and 38.

In an embodiment, the binding agent comprises the sequences set forth in SEQ ID NOs: 36 and 39.

In an embodiment, the binding agent comprises the sequences set forth in SEQ ID NOs: 37 and 39.

In an embodiment, the binding agent comprises the sequences set forth in SEQ ID NOs: 38 and 19.

In an embodiment, the binding agent comprises the sequence set forth in SEQ ID NO: 42.

According to the first aspect, the payload molecule may be any number of agents, including but not limited to cytotoxic agents or drugs such as chemotherapeutic agents, growth inhibitory agents, toxins (for example, an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof) , or a radioactive isotope or a compound comprising a radioactive isotope (that is, a radioconjugate) .

In an embodiment according to the first aspect, the payload molecule is a toxic payload molecule, a fluorescent label molecule or a radioactive molecule. The toxic payload molecule may, in principle, be any toxic molecule suitable for conjugation to the binding agent according to the first aspect. It may be any compound that results in the death of a cell, or induces cell death, or in some manner decreases cell viability. The toxic payload molecule can be any of many small molecule drugs, including, but not limited to, dolastatins ; auristatins ; epothilones ; daunorubicins and doxorubicins; alkylating agents, such as thiotepa and cyclophosphamide (CYTOXAN™) ; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines, such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylene-phosphoramide, triethylenethiophosphaoramide and trimethylolomelamine ; acetogenins (especially bullatacin and bullatacinone) ; camptothecins (including the synthetic analogue topotecan) ; bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues) ; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); duocarmycin (including the synthetic analogues, KW-2189 and CBI- TMI); eleutherobin; pancratistatin; sarcodictyins ; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics, such as the enediyne antibiotics (e.g. calicheamicins , especially calicheamicin γΐ ; dynemicin, including dynemicin A; esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores ) , aclacinomysins , actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin; chromomycins , dactinomycin, detorubicin, 6-diazo-5-oxo-L- norleucine, other doxorubicin derivatives including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino- doxorubicin and deoxydoxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, nitomycins, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; antimetabolites, such as methotrexate and 5-fluorouracil (5-FU) ; folic acid analogues, such as denopterin, methotrexate, pteropterin, trimetrexate ; purine analogs, such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine ; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-fluorouracil ; androgens, such as calusterone, dromostanolone propionate, epitiostanol , mepitiostane, testolactone ; anti-adrenals, such as aminoglutethimide, mitotane, trilostane; folic acid replenisher, such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfomithine; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids , such as maytansine, ansamitocins , DM- 1, DM-4; mitoguazone; mitoxantrone ; mopidamol; nitracrine; pentostatin; phenamet; pirarubicin; podophyllinic acid; 2- ethylhydrazide ; procarbazine; PSK®; razoxane; rhizoxin; sizofuran; spirogermanium; tenuazonic acid; triaziquone; 2 , 2 2 "-trichlorotriethylamine ; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine) ; urethan; vindesine; dacarbazine; mannomustine ; mitobronitol ; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C") ; cyclophosphamide; thiotepa; taxoids, e.g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.) and doxetaxel (TAXOTERE®, Rhone- Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine ; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO) ; retinoic acid; capecitabine ; anti-hormonal agents that act to regulate or inhibit hormone action on tumors, such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4 ( 5 ) -imidazoles , 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston) ; and anti-androgens , such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; siRNA; and pharmaceutically acceptable salts, acids or derivatives of any of the above as well as analogues and derivatives thereof, some of which are described below.

In an embodiment, the toxic payload molecule is a dolastatin, auristatin, doxorubicin, maytansinoid, epirubicin, duocarmycin, or any analogue or derivative thereof.

In an embodiment, the toxic payload molecule is dolastatin 10, dolastatin 15 or any derivative thereof.

In an embodiment, the toxic payload molecule is auristatin F or any derivative thereof.

In an embodiment, the toxic payload molecule is dolastatin 10, dolastatin 15, or auristatin F.

In an embodiment, the toxic payload molecule is dolastatin 10.

In an embodiment, the toxic payload molecule is dolastatin 15.

In an embodiment, the toxic payload molecule is auristatin F.

Dolastatins that can be used in the present invention are well known in the art and can be isolated from natural sources according to known methods or prepared synthetically according to known methods .

Examples of suitable dolastatins include monomethyl and desmethyl dolastatins 10, 15, C, D and H, monomethyl and desmethyl isodolastatin H, and analogues and derivatives thereof. These dolastatins contain a primary or secondary amine at the N-terminus. Dolastatins 10 and 15 are the most potent toxic payload molecules among the naturally occurring dolastatins. Monomethyl and desmethyl dolastatins 10 and 15 can be prepared by chemical synthesis according to standard peptide synthesis chemistry.

Auristatins that can be used in the present invention include (but are not limited to) monomethyl and desmethyl auristatins E, F, EB, EFP, PY, PYE, PE, PHE, TP, 2-AQ and 6-AQ, e.g. described in U.S. Pat. No. 5,635,483; Int. J. Oncol. 15:367-72 (1999); Mol. Cancer Ther. 3:921-32 (2004); U.S. application Ser. No. 11/134,826; U.S. Patent Publication Nos. 20060074008 and 2006022925; and Pettit, G.R., et al . (2011) J. Nat. Prod. 74:962-8.

In an embodiment, the monomethyl or desmethyl dolastatin 15 analogue or derivative is selected from the group consisting of monomethyl and desmethyl dolastatin 15, monomethyl and desmethyl cemadotin, monomethyl and desmethyl tasidotin, and monomethyl and desmethyl P5 (the corresponding dimethyl compounds are described in Bai et al . 2009. Mol. Pharmacol. 75:218-26) .

The toxic payload molecule may also be daunorubicin or doxorubicin. The primary amine group of the daunosamine moiety can be used, or daunorubicin or doxorubicin can be modified to comprise another primary or secondary amine moiety.

In an embodiment, the toxic payload molecule is a maytansinoid .

In an embodiment, the toxic payload molecule is maytansine, an ansamitocin, DM1 or DM4 (also known as DM-4) .

In an embodiment, the toxic payload molecule is DM1. DM1 is also known as DM-1 and mertansine.

In an embodiment, the toxic payload molecule is a rubicin. Suitable rubicins may be e.g. daunorubicins , doxorubicins, detorubicin, other doxorubicin derivatives including morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, deoxydoxorubicin, epirubicin, esorubicin, idarubicin, rodorubicin, zorubicin, and pirarubicin.

In an embodiment, the toxic payload molecule is epirubicin .

In an embodiment, the toxic payload molecule is duocarmycin. Suitable duocarmyxins may be e.g. duocarmycin A, duocarmycin Bl, duocarmycin B2, duocarmycin CI, duocarmycin C2, duocarmycin D, duocarmycin SA, duocarmycin MA, and CC-1065. The term "duocarmycin" should be understood as referring also to synthetic analogs of duocarmycins , such as adozelesin, bizelesin, carzelesin, KW-2189 and CBI-TMI.

In an embodiment, the duocarmycin is a duocarmycin suitable for conjugating to the binding agent according to the first aspect. In an embodiment, the duocarmycin comprises an amino group or another suitable chemical group for conjugating the duocarmycin to the binding agent according to the first aspect. In an embodiment, the amino group is a free amino group.

One skilled in the art of payload molecules will readily understand that each of the payload molecules described in this specification can be modified in such a manner that the resulting compound still retains the specificity and/or activity of the starting compound. The skilled person will also understand that many of these compounds can be used in place of the toxic payload molecules described herein. Thus, the toxic payload molecules of the present invention should be understood as including any analogues and derivatives of the compounds described herein.

In an embodiment, the payload molecule is a toxic payload molecule selected from the group consisting of dolastatin, auristatin, doxorubicin, maytansinoid, epirubicin, duocarmycin, and any analogue or derivative thereof.

In this context, the term "fluorescent label molecule" may refer to any fluorophore which can re-emit light upon light excitation. Such a fluorescent label molecule may be suitable for detection, visualization and/or quantitation. Suitable fluorescent label molecules may be e.g. FITC, TRITC, and the Alexa and Cy dyes.

In this context, the term "radioactive molecule" may refer to any molecule that comprises a radioactive atom. The term "radioactive molecule" may refer to a radioactive molecule suitable for radiation therapy. It may also refer to a radioactive molecule suitable for detection, visualization and/or quantitation.

The payload molecule may be selected so that it comprises a functional group that allows for conjugating it to the binding agent according to the first aspect. The payload molecule may also be modified prior to reacting it with other components of the binding agent according to the first aspect. In an embodiment, the payload molecule comprises a primary or secondary amine moiety. In an embodiment, the payload molecule is modified to comprise a primary or secondary amine moiety. In an embodiment, the amine-modified payload molecule essentially retains the activity of the original payload molecule after conj ugation . In an embodiment, the payload molecule is covalently linked to the binding agent.

In an embodiment, the payload molecule is covalently linked to the first binding component.

In an embodiment, the payload molecule is covalently linked to the second binding component.

The payload molecule may be covalently linked to the binding agent, to the first binding component and/or to the second binding component directly, via a bond, or indirectly, e.g. via a linker or linker group. An overview of the technology of covalently linking or conjugating payload molecules to binding agents is provided in Ducry et al . , Bioconjugate Chem. , 21:5-13 (2010), Carter et al . , Cancer J. 14(3) :154 (2008) and Senter, Current Opin. Chem. Biol. 13:235-244 (2009). Linkers that may, in principle, be utilised are described e.g. in Dosio et al . , Toxins 2011, 3, 848-883, and Sammet et al . , Pharm. Pat. Analyst 2012, 1(1), 2046-8954.

The linker may be e.g. a peptide linkage or a linker cleavable by a lysosomal hydrolase. As will be appreciated by those in the art, the number of payload molecules, i.e. the drug moieties per binding agent may vary.

In an embodiment, the binding agent comprises a molecule represented by formula II [D-L] _n -P

Formula II wherein P is the first binding component or the second binding component;

n is at least 1 ;

L is a linker group covalently joining P to D; and

D is the payload molecule.

In an embodiment, the binding agent is a molecule represented by formula II.

In an embodiment, the binding agent consists of a molecule represented by formula II.

In an embodiment, n is an integer from 1 to 20. In an embodiment, n is 2-18. In an embodiment, n is 2-16. In an embodiment, n is 2-10. In other embodiments, n is 2-6; 2-5; 2- 4; 2-3; 3-4; or 1, 2, 3 or 4.

In an embodiment, the binding agent comprises a molecule represented by formula III

[D-L-G] _n -P

Formula III wherein P is the first binding component or the second binding component comprising an N-glycan, wherein the N-glycan comprises a GlcNAc residue bound by a β-Ν linkage to an asparagine ;

n is at least 1;

D is the payload molecule;

L is a linker group covalently joining G to D; and

G is a saccharide structure represented by formula IV

Formula IV wherein

R is a glycosidic bond to the N-glycan or a glycosidic bond to the GlcNAc residue bound by a β-Ν linkage to an asparagine ;

X ¹ is H or carboxyl;

X ² , X ³ and X ⁴ are each independently OH, H, amino, C2-C6 acylamide, phosphate or sulphate ester, or a bond to L;

X ⁵ is CH ₂ OH, carboxyl, CH ₃ , H, C ₁ -C3 alkyl or substituted C ₁ -C3 alkyl, or a bond to L;

with the proviso that one substituent selected from X ² , X ³ , X ⁴ and X ⁵ is a bond to L or bonded via a bond to L; and

with the proviso that when X ¹ is carboxyl, then X ² is H, X ³ is OH, X ⁵ is C ₁ -C3 alkyl or substituted C ₁ -C3 alkyl; R is a glycosidic bond to the N-glycan; and X ⁴ is a bond to L or X ⁵ is bonded via a bond to L; or when X ¹ is H, then R is a glycosidic bond to the N- glycan or to the GlcNAc residue bound by a β-Ν linkage to an asparagine .

In an embodiment, the binding agent is a molecule represented by formula III.

In an embodiment, the binding agent consists of a molecule represented by formula III.

These embodiments have the added utility that they comprise a relatively small payload molecule-glycan moiety that is efficiently released inside cells. This may be particularly beneficial when the payload molecule is a toxic payload molecule. Further, the moiety released is relatively small; small toxic payload molecule conjugates tend to be more toxic than large toxic payload molecule conjugates e.g. comprising a complex-type N-glycan core structure. The toxic payload molecule-glycan conjugate released from the binding agent in cells is capable of delivering the toxic payload molecule into cells and further into the cytosol, the nucleus or the endoplasmic reticulum. The binding agent is also sufficiently stable towards chemical or biochemical degradation during manufacturing or in physiological conditions, e.g. in blood, serum, plasma or tissues, and in reducing conditions, in low pH and inside cells, cellular organelles, endosomes and lysosomes.

In an embodiment, n is an integer from 1 to 20. In an embodiment, n is 2-18. In an embodiment, n is 2-16. In an embodiment, n is 2-10. In other embodiments, n is 2-6; 2-5; 2-4; 2-3; 3-4; or 1, 2, 3 or 4.

In an embodiment, G is a saccharide structure represented by formula V

Formula V wherein

R is a glycosidic bond to the N-glycan; X ⁴ is OH, H, amino, C ₂ ^_ C6 acylamide, phosphate or sulphate ester, or a bond to L;

X ⁵ is C ₁ -C3 alkyl or substituted C ₁ -C3 alkyl;

and X ⁴ is a bond to L or X ⁵ is bonded via a bond to L.

In an embodiment, the N-glycan comprises a terminal Θβΐβ residue and R is a glycosidic bond to the terminal Θβΐβ residue .

In an embodiment, the N-glycan consists of the structure represented by formula VI

(Fuc 6),

\

GicNAc{i5-N-Asn)

Formula VI wherein (β-Ν-Asn) is a β-Ν linkage to an asparagine and y is 0 or 1;

X ¹ is H;

X ² , X ³ and X ⁴ are each independently OH, H, amino, C2-C6 acylamide, phosphate or sulphate ester, or a bond to L;

X ⁵ is CH ₂ OH, carboxyl, CH ₃ , H, C ₁ -C3 alkyl or substituted C ₁ -C3 alkyl, or a bond to L;

with the proviso that one substituent selected from X ² , X ³ , X ⁴ and X ⁵ is a bond to L or bonded via a bond to L; and

R is a glycosidic bond to the GlcNAc residue.

In an embodiment, the linker group is hydrophilic.

In an embodiment, the linker group comprises at least one OH group.

In an embodiment, the linker group comprises at least one moiety derived from one or more saccharide units.

In an embodiment, the linker group is represented by formula VII

Formula VII wherein

Y is an oxygen, sulphur, amine, amide, peptide or absent, wherein the peptide is an E ₁ -P-E ₂ unit in which Ei and E ₂ are independently C=0, 0 or NR _P , wherein R _p is H, C ₁ -C6 alkyl or substituted C ₁ -C6 alkyl, P is a peptide unit from 2 to 5 amino acids in length, and Ei and E ₂ can independently be linked to the peptide through the terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide;

Z is a saccharide or absent;

D' is the payload molecule, wherein the payload molecule comprises an amine moiety, through which the payload molecule is bound so as to form a secondary or tertiary amine;

Ri, R2, R3, R4, R5, R7, ¾ and R9 are each independently H, OH, amine, C ₂ -C6 acylamide, carboxyl, substituted carboxyl, C1-C6 alkyl or substituted C1-C6 alkyl;

W is H, CH ₂ OH, CH ₃ , carboxyl, substituted carboxyl, C ₁ -C6 alkyl or substituted C ₁ -C6 alkyl;

a is an integer from 0 to 6;

b is 0 or 1;

c and e are each independently an integer from 0 to 7; d is an integer from 1 to 7;

Q is E'-F'-E, wherein F' is an amine, amide, disulfide, thioether, thioester, hydrazone, Schiff base, oxime, olefin metathesis reaction product, triazole or phosphine group, or other group generated by the reaction of the functional group F- E and the functional group F' , wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and F' is an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine; and E is absent or a polyethyleneoxy unit of formula (CH ₂ CH ₂ 0) _P , wherein p is an integer from 2 to about 20; and E and E' are each independently absent or a polyethyleneoxy unit of formula (CH ₂ CH ₂ 0) _P , wherein p is an integer from 2 to about 20; and

Q is bound via a bond to G.

This embodiment has the added utility that the linker group conveys good solubility in aqueous solutions. In an embodiment, L is a linker group represented formula VIII

wherein

Y is an oxygen, sulphur, amine, amide, peptide or absent, wherein the peptide is an E ₁ -P-E ₂ unit in which Ei and E ₂ are independently C=0, 0 or NR _P , wherein R _p is H, C ₁ -C6 alkyl or substituted C ₁ -C6 alkyl, P is a peptide unit from 2 to 5 amino acids in length, and Ei and E ₂ can independently be linked to the peptide through the terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide;

Z is a saccharide or absent;

D' is the payload molecule, wherein the payload molecule comprises an amine moiety, through which the payload molecule is bound so as to form a secondary or tertiary amine;

Ri, R ₂ , R9 and Rio are each independently H, OH, amine,

C2-C6 acylamide, carboxyl, substituted carboxyl, C1-C6 alkyl or substituted C1-C6 alkyl;

a is an integer from 0 to 6;

e is an integer from 0 to 3;

d and f are integers from 0 to 4 with the proviso that their sum is from 1 to 4;

Q is E'-F'-E, wherein F' is an amine, amide, disulfide, thioether, thioester, hydrazone, Schiff base, oxime, olefin metathesis reaction product, triazole or phosphine group, or other group generated by the reaction of the functional group F- E and the functional group F' , wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and F' is an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine ; and E is absent or a polyethyleneoxy unit of formula ( CH2CH20 ) _P , wherein p is an integer from 2 to about 20; and E and E' are each independently absent or a polyethyleneoxy unit of formula ( CH2CH20 ) _P , wherein p is an integer from 2 to about 20; and

Q is bound via a bond to G.

In an embodiment, L is a linker group represented by formula IX

Formula IX wherein

Y is an oxygen, sulphur, amine, amide, peptide or absent, wherein the peptide is an E ₁ -P-E ₂ unit in which Ei and E ₂ are independently C=0, 0 or NR _P , wherein R _p is H, C ₁ -C6 alkyl or substituted C ₁ -C6 alkyl, P is a peptide unit from 2 to 5 amino acids in length, and Ei and E ₂ can independently be linked to the peptide through the terminal nitrogen, terminal carbon or through a side chain of one of the amino acids of the peptide;

Z is a saccharide or absent;

D' is the payload molecule, wherein the toxic payload molecule comprises an amine moiety, through which the toxic payload molecule is bound so as to form a secondary or tertiary amine;

Ri and R ₂ are each independently H, OH, amine, C ₂ -C6 acylamide, carboxyl, substituted carboxyl, C ₁ -C6 alkyl or substituted C1-C6 alkyl;

a is an integer from 0 to 6;

c and e are each independently an integer from 0 to 3;

Q is E'-F'-E, wherein F' is an amine, amide, disulfide, thioether, thioester, hydrazone, Schiff base, oxime, olefin metathesis reaction product, triazole or phosphine group, or other group generated by the reaction of the functional group F- E and the functional group F' , wherein F is a functional group that can react with an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine, and F' is an amine, thiol, azide, alkene, alkyne, aldehyde, ketone, carboxylic acid or hydroxylamine; and E is absent or a polyethyleneoxy unit of formula ( CH2CH20 ) _P , wherein p is an integer from 2 to about 20; and E and E' are each independently absent or a polyethyleneoxy unit of formula ( CH2CH20 ) _P , wherein p is an integer from 2 to about 20; and

Q is bound via a bond to G.

In an embodiment of the invention, F is an amine reacting group, a thiol reactive group, an azide reactive group, an alkyne reactive group, a carbonyl reactive group or a hydroxylamine reactive group.

In an embodiment of the invention, F is an amine reacting group, such as (but not limited) to an N- hydroxysuccinmide ester, p-nitrophenyl ester, dinitrophenyl ester, or pentafluorophenyl ester.

In an embodiment of the invention, F is a thiol reactive group, such as (but not limited to) pyridyldisulfide, nitropyridyldisulfide, maleimide, haloacetate or carboxylic acid chloride.

In an embodiment of the invention, F is an azide reactive group, such as (but not limited to) alkyne.

In an embodiment, F is an alkyne.

In an embodiment, F is CH≡C .

In an embodiment, F is a dibenzocyclooctyl group

(DBCO) .

In an embodiment of the invention, F is an alkyne reactive group, such as (but not limited to) azide.

In an embodiment, F is azide.

In an embodiment of the invention, F is a carbonyl reactive group, such as (but not limited to) hydroxylamine.

In an embodiment of the invention, F is a hydroxylamine reactive group, such as (but not limited to) aldehyde or ketone.

In an embodiment of the invention, F is isothiocyanate, isocyanate, sulfonyl chloride, glyoxal, epoxide, oxirane, carbonate, aryl halide, imidoester, carbodiimide, or anhydride.

In an embodiment, Z is absent.

In an embodiment, Z is a saccharide. In an embodiment, Z is an oligosaccharide with a degree of polymerization from 1 to about 20; from 1 to 10; from 1 to 8; from 1 to 6; from 1 to 5; from 1 to 4; from 1 to 3; from 1 to 2; or 1, 2, 3, 4 or 5.

In an embodiment, Z is a monosaccharide, disaccharide or trisaccharide .

In an embodiment, Z is OH

In an embodiment, Z is H.

In an embodiment, a is 1, 2 , 3 , 4 , 5 , or 6.

In an embodiment, a is 1.

In an embodiment, b is 0.

In an embodiment, b is 1.

In an embodiment, c is 0.

In an embodiment, c is 1, 2, 3, 4, 5, 6 or 7.

In an embodiment, d is 1, 2, 3, 4, 5, 6 or 7.

In an embodiment, d is 3, 4 or 5.

In an embodiment, d is 3.

In an embodiment, d is 4.

In an embodiment, d is 5.

In an embodiment, d is 6.

In an embodiment, e is 0.

In an embodiment, e is 1, 2, 3, 4, 5, 6 or 7.

In an embodiment, d is 3; and R ₇ is H.

In an embodiment, d is 4; and R ₇ is H.

In an embodiment, b is 1; and R3 and R4 are each

In an embodiment, a is 1; and Ri and R2 are each

In an embodiment, e is 1; and R8 and R9 are each

In an embodiment, a, b, c , or e is 0.

In an embodiment, a, b, c , and/or e is 0.

In an embodiment, W is H.

In an embodiment, a is 2 or 3; and Ri and R2 are

In an embodiment, Y is oxygen .

In an embodiment, Y is sulphur .

In an embodiment, Y is a peptide .

In ar embodiment according to the first or second aspect, the binding agent is preferentially internalized by a tumor cell or a cancer cell. In an embodiment, the binding agent is preferentially internalized by a tumor cell or a cancer cell as compared to a non-tumor cell or a non-cancer cell.

The present invention further relates to a pharmaceutical composition comprising the binding agent according to one or more embodiments of the first or second aspect .

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. Examples of suitable pharmaceutically acceptable carriers are well known in the art and may include e.g. phosphate buffered saline solutions, water, oil/water emulsions, wetting agents, and liposomes. Compositions comprising such carriers may be formulated by methods well known in the art. The pharmaceutical composition may further comprise other components such as vehicles, additives, preservatives, other pharmaceutical compositions administrated concurrently, and the like.

In an embodiment, the pharmaceutical composition comprises an effective amount of the binding agent according to one or more embodiments of the first or second aspect.

In an embodiment, the pharmaceutical composition comprises a therapeutically effective amount of the binding agent according to one or more embodiments of the first or second aspect.

The term "therapeutically effective amount" or

"effective amount" of the conjugate should be un-derstood as referring to the dosage regimen for modulating the growth of cancer cells and/or treating a patient's disease. The therapeutically effective amount may be selected in accordance with a variety of factors, including the age, weight, sex, diet and medical condition of the patient, the severity of the disease, and pharmacological considerations, such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular conjugate used. The therapeutically effective amount can also be determined by reference to standard medical texts, such as the Physicians Desk Reference 2004. The patient may be male or female, and may be an infant, child or adult.

The term "treatment" or "treat" is used in the conventional sense and means attending to, caring for and nursing a patient with the aim of combating, reducing, attenuating or alleviating an illness or health abnormality and improving the living conditions impaired by this illness, such as, for example, with a cancer disease.

In an embodiment, the pharmaceutical composition comprises a composition for e.g. oral, parenteral, transdermal, intraluminal, intraarterial, intrathecal and/or intranasal administration or for direct injection into tissue. Administration of the pharmaceutical composition may be effected in different ways, e.g. by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration .

According to a third aspect of the invention there is provided a binding agent, wherein the binding agent comprises a sequence selected from the group consisting of SEQ ID NO: 17, 36, 37, and 38.

According to a fourth aspect of the invention there is provided a binding agent, wherein the binding agent comprises sequences set forth in SEQ ID NOs : 17 and SEQ ID NO: 12; or SEQ ID NOs: 17 and SEQ ID NO: 14; or SEQ ID NOs: 36 and 39; or SEQ ID NOs: 37 and 39; or or SEQ ID NOs: 38 and 19.

In an embodiment according to the third or fourth aspect, the binding agent comprises a payload molecule. In the context of this aspect, the payload molecule may be any payload molecule described in this specification.

The present invention further relates to a pharmaceutical composition comprising the binding agent according to one or more embodiments of third or fourth aspect.

The present invention further relates to a nucleic acid encoding the protein moiety of the binding agent according to one or more embodiments of the first, second, third or fourth aspect .

The present invention further relates to an isolated nucleic acid encoding the protein moiety of the binding agent according to one or more embodiments of the first, second, third or fourth aspect.

The isolated nucleic acid comprises a nucleotide sequence encoding the protein moiety of a binding agent according to the first, second, third or fourth aspect. In the context of this specification, the term

"protein moiety" should be understood as referring to at least one polypeptide, wherein the polypeptide comprises the first binding component, the second binding component or both. The protein moiety may also be a fusion polypeptide comprising both the first binding component and the second binding component.

The present invention further relates to a recombinant expression vector comprising the nucleic acid according one or more embodiments of the invention.

The recombinant expression vector according to one or more embodiments of the present invention may be e.g. a plasmid, phagemid, phage or viral vector, into which a nucleic acid encoding a binding agent according to one or more embodiments of the first, second, third or fourth aspect is inserted.

The nucleic acid and the recombinant expression vector may be produced by variety of techniques such as chemically synthesized using, for example, synthesizers. Recombinant expression vectors of the invention may be capable of expressing the RNA and/or protein products of the nucleic acids (DNA) . The recombinant expression vector may further comprise regulatory sequences, including a promoter operably linked to the open reading frame (ORF) . The vector may further comprise a selectable marker sequence. Specific initiation and secretory signals also may be included for efficient translation of inserted target gene coding sequences.

The recombinant expression vector may be advantageously selected and constructed depending upon the use intended for the binding agent being expressed. For example, when a large quantity of a binding agent is to be produced, for the generation of antibodies, for example, vectors which direct the expression of high levels of protein products that are readily purified may be desirable.

The present invention further relates to a host cell comprising the recombinant expression vector according to one or more embodiments of the invention.

In an embodiment, the host cell is an isolated host cell .

The host cell can be virtually any cell for which recombinant expression vectors are available. It may be, for example, a higher eukaryotic host cell, such as a mammalian cell, a lower eukaryotic host cell, such as a yeast cell or a fungal cell, and may be a prokaryotic cell, such as a bacterial cell, for example E. coli, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus .

In an embodiment, the mammalian cell is a CHO cell, cell line CHO-K1 (ATCC CCL-61), cell line DUXB11 (ATCC CRL-9096) and cell line Pro-5 (ATCC CRL-1781) registered at ATCC, CHO-S (Cat # 11619 of Life Technologies)), a BHK cell (ATCC accession no. CCL 10), a NSO cell, NSO cell line (RCB 0213) registered at RIKEN Cell Bank, The Institute of Physical and Chemical Research, a SP2/0 cell, a SP2/0-Agl4 cell, SP2/0-Agl4 cell (ATCC CRL-1581), a YB2/0 cell, a PER cell, a PER . C6 cell, a rat myeloma cell line YB2 /3HL . P2. Gl 1.16Ag .20 cell (including cell lines established from Y3/Agl.2.3 cell (ATCC CRL-1631), YB2/3HL.P2.G11.16Ag.20 cell, YB2 /3HL . P2. Gl 1.16Ag .20 cell (ATCC CRL-1662), and any sub-lines obtained by naturalizing these cell lines to media in which they can grow, and the like) , a hybridoma cell, a human leukemic Namalwa cell, or an embryonic stem cell. Introduction of the recombinant construct into the host cell can be effected, for example, by calcium phosphate transfection, DEAE, dextran mediated transfection, electroporation or phage infection.

The present invention further relates to a method for producing the binding agent according to one or more embodiments of the first, second, third or fourth aspect, comprising

culturing the host cell according to one or more embodiments of the invention under conditions suitable for expression of the nucleic acid encoding the protein moiety of a binding agent according to one or more embodiments of the first, second, third or fourth aspect to produce the protein moiety of the binding agent; and

isolating the protein moiety of the binding agent from the culture.

In an embodiment according to the first aspect, the method comprises conjugating a payload molecule to the protein moiety of the binding agent according to one or more embodiments of the first, second, third or fourth aspect. The present invention further relates to the binding agent according one or more embodiments of the first, second, third or fourth aspect or the pharmaceutical composition according to according one or more embodiments of the invention for use as a medicament.

The present invention further relates to the binding agent according one or more embodiments of the first, second, third or fourth aspect or the pharmaceutical composition according to according one or more embodiments of the invention for use in the treatment of cancer, autoimmune disease, inflammatory disorder or infection.

The present invention further relates to the binding agent according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention for use in the treatment of a disorder selected from the group consisting of cancer, autoimmune disease, inflammatory disorder and infection.

The present invention further relates to the binding agent according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention for use in the treatment of cancer.

In an embodiment, the cancer is a leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cancer, small-cell lung cancer, head-and-neck cancer, multidrug resistant cancer, glioma, melanoma and testicular cancer.

The present invention further relates to the use of the binding agent according one or more embodiments of the first or second aspect or the pharmaceutical composition according to one or more embodiments of the invention in the manufacture of a medicament .

The present invention further relates to the use of the binding agent according one or more embodiments of the first or second aspect or the pharmaceutical composition according to one or more embodiments of the present invention in the manufacture of a medicament for the treatment of cancer, autoimmune disease, inflammatory disorder or infection. The present invention further relates to the use of the binding agent according one or more embodiments of the first or second aspect or the pharmaceutical composition according to one or more embodiments of the present invention in the manufacture of a medicament for the treatment of a disorder selected from the group consisting of cancer, autoimmune disease, inflammatory disorder and infection.

The present invention further relates to a method of treating cancer, autoimmune disease, inflammatory disorder or infection, wherein the binding agent according to according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention is administered to a human in an effective amount.

The present invention further relates to a method of treating a disorder selected from the group consisting of cancer, autoimmune disease, inflammatory disorder and infection, wherein the binding agent according to according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention is administered to a human in an effective amount.

The present invention further relates to a method of treating cancer, wherein the binding agent according to according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention is administered to a human in an effective amount.

In an embodiment, the cancer is a leukemia, lymphoma, breast cancer, prostate cancer, ovarian cancer, colorectal cancer, gastric cancer, squamous cancer, small-cell lung cancer, head-and-neck cancer, multidrug resistant cancer, glioma, melanoma and testicular cancer.

The present invention further relates to a method for modulating growth of a cell population, wherein the method comprises contacting the binding agent according to according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention with the cell population. In an embodiment, the cell population is a tumor cell population or a cancer cell population.

The present invention further relates to a method of treating and/or modulating the growth of and/or prophylaxis of tumor cells in humans or animals, wherein the binding agent according to according one or more embodiments of the first or second aspect or the pharmaceutical composition according to according one or more embodiments of the invention is administered to a human or animal in an effective amount.

In an embodiment, the tumor cells are selected from the group consisting of leukemia cells, lymphoma cells, breast cancer cells, prostate cancer cells, ovarian cancer cells, colorectal cancer cells, gastric cancer cells, squamous cancer cells, small-cell lung cancer cells, head-and-neck cancer cells, multidrug resistant cancer cells, and testicular cancer cells.

The embodiments of the invention described hereinbefore may be used in any combination with each other. Several of the embodiments may be combined together to form a further embodiment of the invention. A product, a use or a method to which the invention is related may comprise at least one of the embodiments of the invention described hereinbefore.

The binding according to one or more embodiments of the first or second aspect has a number of advantageous properties.

The binding agent is preferentially internalized by tumor or cancer cells. The binding agent has a good internalization efficiency.

The presence of two binding components improves capability and specificity of the binding of the binding agent to tumor or cancer cells.

The binding agent according to the first aspect allows for conjugating a relatively large number of payload molecules to the binding agent. The binding agent has low immunogenicity; yet it is well soluble in aqueous solutions.

EXAMPLES

In the following, the present invention will be described in more detail. Reference will now be made in detail to the embodiments of the present invention, examples of which are illustrated in the accompanying drawings. The description below discloses some embodiments of the invention in such detail that a person skilled in the art is able to utilize the invention based on the disclosure. Not all steps of the embodiments are discussed in detail, as many of the steps will be obvious for the person skilled in the art based on this specification .

Example 1. 2G12 antibodies and toxin conjugates

2G12-antibody (human IgG; Polymun Scientific, Austria) was subjected to galactosylation and sialylation using UDP- galactose and CMP- 9-azido-N-acetyl-neuraminic as in Example 5. Fc-fragments were released from a small aliquot of reaction mixture with Fabricator enzyme and recovered using Poros Rl tips. MALDI-TOF MS analysis of purified Fc revealed major signal at m/z 25873 corresponding to Fc with 9-azido-NeuNAc-G2F .

9-azido-sialylated antibody was purified with Protein G as in Example 5 and DM1-DBCO was conjugated to 9-azido- sialylated 2G12 as in Example 5. Fabricator analysis of conjugated antibody was done as described below. MALDI-TOF MS of purified Fc revealed major signal at m/z 26927 corresponding to Fc chain carrying DMl-DBCO-9-azido-NeuNAc-G2F. A minor signal was detected at m/z 28299 corresponding to (DMl-DBCO-9-azido- NeuNAc) 2-G2F . Based on the peak intensities, approximately 2.3 toxins were conjugated to 2G12 antibody.

Synthesis of 2G12-drug conjugates by copper (I) catalyzed click reaction

To 300 yg (2 nmol) of 2G12 antibody in PBS - 5 % mannitol - 0.1 % Tween20 solution (80 μΐ) was added lOx molar excess of NHS-PEG ₄ -N ₃ dissolved in DMSO (1 or 4 μΐ) and the reaction was allowed to proceed for 3 hours at room temperature.

To minimize loss of antibody, the reaction was conducted in an

Amicon Ultra centrifugal filter unit (30K, 0.5 ml) . Low molecular weight reagents were then removed by Amicon centrifugal filter with repeated addition of PBS - 5 % mannitol

- 0.1 % Tween20.

To prepare 2G12-drug conjugate (Scheme 1) , 50x molar excess of TGTA and sodium ascorbate in ¾0 (2 μΐ) , lOx molar excess of CU S C in ¾0 (0.34 μΐ, final concentration 500 μΜ) and 20x molar excess of N- ( 6-propargyl-D-galactosyl ) -dolastatin 10 in DMSO (4 μΐ) were added into the solution of 2 nmol of 2G12- PEG ₄ -N3 (40 μΐ) . The reactions were performed at room temperature for 45 minutes. The conjugated antibodies were purified by Amicon centrifugal filter as above.

The DAR of the 2G12-drug conjugate was estimated to be about 2, by isolating the Fc-fragments and light chains followed by MALDI-TOF MS analysis.

Scheme 1. Structure of (N-galactosyl ) -Modo-triazole-PEG-2G12 conjugate.

2G12 variantl

2G12 light chain encoding sequence is ordered as a synthetic gene from a supplier and cloned into e.g. a CHO expression vector.

2G12 heavy chain variantl is assembled by overlap PCR from two sequences. Sequence encoding amino acids 1-220 is ordered as a synthetic gene from a supplier, sequence encoding amino acids 221-453 is PCR amplified from GCM012 expression vector.

For transient expression, subsequent cloning of light and heavy chains are made as described with GCM005 below, and for stable expression as described for GCM023 below. 2G12 variant2 (2G12 variantl+I19N)

Heavy chain mutation I19N is generated to 2G12 variantl backbone by PCR overlap, using overlapping oligos containing the mutated site. Full length heavy chain is assembled from two pieces essentially as described for GCM023. GCM020 (2G12 variantl+I19N+F78S)

Heavy chain mutation F78S is generated to 2G12 variantl or 2G12 variant2 heavy chain backbone by PCR overlap, using overlapping oligos containing the mutated site. Full length heavy chain is assembled from two pieces essentially as described for GCM023.

EXAMPLE 2. Generation of GCM005 (cetuximab heavy chain fused to 2G12 scFv)

Cetuximab light chain fragment was amplified by PCR from a plasmid having a CHO codon optimized light chain DNA sequence using primers enabling cloning into the transient expression vector pCEP4.

Cetuximab heavy chain and 2G12 scFv fragments were PCR amplified separately (cetuximab heavy chain from a plasmid having CHO codon optimized heavy chain; 2G12 scFv from a plasmid having CHO codon optimized 2G12 scFv) . PCR fragments were purified from agarose gel and joined by overlap extension PCR. The whole 2220 bp join product was amplified, cut with restriction enzymes, and ligated to transient expression vector pCEP4. All antibodies produced in the Examples used signal peptides of SEQ ID NO: 40 (for heavy chains) or SEQ ID NO: 41 (for light chains) .

Transient transfection and production of GCM005 antibody in CHO- s cells

Transfection was carried out according to Freedom CHO-S Kit protocol (Life Technologies) . 22,5 μg of Cetuximab light chain containing plasmid and 22,5 μg of cetuximab heavy chain + 2G12 scFV containing plasmid was used for transfection of 30 x 10 ^s CHO-S cells. After 4 days of culture supernatant was collected by pelleting cells at 350 x g for 5 minutes at room temperature and the antibody was purified from culture supernatant by Protein A column (High Trap MAb Select Sure; 5 ml; GE Healthcare) and AKTA purifier instrument (GE Healthcare) as described in purification of GCM023. Synthesis of GCM005-drug conjugate by copper (I) catalyzed click reaction

Synthesis of 6-0-propargyl-D-galactose

Scheme 2. Synthesis of 6-0-propargyl-D-galactose. i) NaH, propargyl bromide, DMF, RT, 3 h, 91%; ii) 60% TFA, 50°C, 1 h, quantitative.

1 , 2 ; 3 , 4-di-O-isopropylidene- 6-O-propargyl- -D- galactopyranose (compound 3 in Scheme 2) . To a solution containing 0.27g (l.Ommol) compound 1 in 5ml dry DMF (under an argon atmosphere) was added 75mg (2.0 equiv.) NaH at 0°C. The resulting mixture was stirred for 20 min. and 171μ1 (1.5 equiv.) of propargyl bromide was added. After 20 min. the mixture was brought to RT and stirred for an additional 2.5 hours. The mixture was cooled on an ice bath and quenched by the addition of MeOH (0.5ml) . The reaction mixture was brought to RT, diluted with 20ml CH2C I 2 and washed with 20ml saturated NaHC0 ₃ -solution . The water phase was extracted with 20ml CH2C I 2 . The combined organic phase was washed with 20ml ¾0, dried over a ₂ S0 ₄ , filtered and concentrated. The crude product was purified by column chromatography (Hexane : EtOAc 2:1) to give compound 2 as a white solid (0.27g, 91%). TLC: R _f = 0.77 (Hexane : EtOAc 1:1). ^X H NMR (600 MHz, CDC1 ₃ , 22° C ) : δ = 5.54 (d, 1 H, Ji , ₂ = 5.1 Hz, H-l), 4.61 (dd, 1 H, J ₃ , 2 = 2.5, J ₃ , ₄ = 8.0 Hz, H-3), 4.32 (dd, 1 H, H- 2), 4.26 (dd, 1 H, J ₄ , ₅ = 1.9 Hz, H-4), 4.25 (dd, 1 H, J _CH 2a, _≡ cH = 2.4, J _C H2a,cH2b = "15.9 Hz, C¾ _a C≡CH), 4.20 (dd, 1 H, J _CH 2b, _≡ cH = 2.4 Hz, CH2bC≡CH) , 4.00 (ddd, 1 H, J ₅ , _6a = 5.4, J ₅ , ₆ b = 7.1 Hz, H-5), 3.78 (dd, 1 H, J _6a ,6b = -10.1 Hz, H-6a), 3.67 (dd, 1 H, H-6b) , 2.43 (dd, 1 H, CH2C≡CH) , 1.55, 1.45, 1.34 and 1.33 (each s, each 3 H, 0 ₂ C(CH ₃ ) ₂ ) ppm. Synthesis of 6-0- propargyl-D-galactose (Scheme 2, compound 3) . 25mg (0.08mmol) of compound 2 was dissolved in 3ml 60% TFA and the resulting mixture was stirred at 50°C for 1 hour. The mixture was then diluted with water and concentrated to give 6-O-propargyl-D-galactose as a colorless oil (18mg, quantitative, furanose : pyranose 3:97, alpha _P y _ran ose : beta _P y _ran ose 35:65). Selected NMR-data: ^X H NMR (600 MHz, D ₂ 0, 22°C): δ = 5.26 (d, 1 H, Ji, ₂ = 4.7 Hz, H-l _furanose ) , 5.23 (d, 1 H, Ji, ₂ = 3.8 Hz, H- la _pyra nose) , 5.20 (d, 1 H, Ji, ₂ = 3.5 Hz, H-lfuranose ), 4.55 (d, 1 H, Ji , ₂ = 7.9 Hz, Η-1β _{ργΓ¾η03θ} ) .

Synthesis of N- (6-O-propargyl-D-galactosyl) -dolastatin 10

Sodium cyanoborohydride (200ymol) and 6-O-propargyl-D- galactose (45ymol) were added to the solution of monomethyldolastatin 10 (2.5ymol) (Concortis Biosystems) in DMSO (0.7ml) . The mixture was stirred at 60°C for three days. The title compound was isolated by reversed-phase chromatography on a column of Gemini-NX-5u C-18 (Phenomenex) . Synthesis of Cu(I) chelator TGTA (tris { [1- (6-D-galactosyl) -1H- 1 , 2, 3-triazol-4-ylJmethyl}amine)

Scheme 3. i) Tripropargylamine, CUSO4, sodium ascorbate, DMF:H ₂ 0 3:1, RT, 40h, quantitative; ii) 60% TFA H ₂ 0) , 60°C, 2.5h, quantitative. Protected TGTA (2 in Scheme 3) : To a solution containing 43mg of 1 (0.15mmol, 5 equiv.) and 4.3μ1 tripropargylamine (0.03mmol, 1 equiv.) in 2ml of DMF:H20 (3:1) was added 2.4mg CuS0 ₄ (0.015mmol, 0.5 equiv.) and 6.4mg sodium L- ascorbate (0.03mmol, 1 equiv.) . The resulting mixture was stirred at RT for 40h (during this time a white solid precipitated from the reaction mixture) . After 40h, the reaction mixture was diluted with 20ml EtOAc transferred to a separatory funnel and washed with 5ml NH ₄ Cl-solut ion (prepared by dissolving a saturated NH ₄ Cl-solution with equal amount of water 1:1 v/v) and 15ml brine. The organic phase was dried with Na ₂ S0 ₄ , filtered and concentrated to give the crude product. The crude product was purified by column chromatography (EtOAc→EtOAc : MeOH 3:1) to give 2 as a colorless oil (30mg, quantitative) . TLC: R _f = 0.22 (EtOAc) . ^X H NMR (600MHz, CDCI ₃ , 25°C) : δ = 8.56 (s, 3 H, triazole-H) , 5.48 (d, 3 H, Ji, ₂ = 5.0Hz, H-l), 4.67 (dd, 3 H, J _6a , ₅ = 3.1, J _6a ,6b = 14.1Hz, H-6a), 4.65 (dd, 3 H, J ₃ , ₂ = 2.5, J ₃ , ₄ = 8.1, H-3), 4.58 (dd, 3 H, J _6b , ₅ = 9.0Hz, H-6b) , 4.41 and 4.33 (each d, each 3 H. J _NC H2a,NCH2b = 14.1Hz, N(C¾) ₃ ), 4.32 (dd, 3 H, H-2), 4.25 (dd, 3 H, J ₄ ,5 = 1.4Hz, H-4), 4.17 (ddd, 3 H, H-5) , 1.50, 1.39, 1.37 and 1.25 (each s, each 9 H, 0 ₂ C(C¾) ₂ ) ppm. HRMS : calcd. for C ₄₅ H ₆₆ ioOi ₅ a [M+Na] ⁺ 1009.46; found 1009.40.

TGTA (3) : 33mg of 2 (0.034mmol) was dissolved in 3ml 60% TFA (in H ₂ 0) and stirred at 50°C for 1.5 hours. The reaction mixture was then diluted with water, concentrated and dried under vacuum to give 3 as a white solid (25mg, quantitative, :β 2:3) . Selected NMR-data; ^X H NMR (600MHz, D ₂ 0, 25°C) : δ = 8.32 (s, 6 H (a and β, 3 H each), triazole-H), 5.21 (d, 3 H, J _1/2 = 3.9Hz, H-lo , 4.59 (s, 12 H (a and β , 6 H each), N(C¾) ₃ ), 4.50 (d, 3 H, Ji, 2 = 8.1Hz, H-Ιβ) . HRMS: calcd. for C ₂ 7H _{42 10} Oi ₅ a [M+Na] ⁺ 769.27; found 769.23.

Synthesis of GCM005-drug conjugate

To 100 yg of GCM005 antibody in PBS - 5 % mannitol - 0.1 % Tween20 solution (150 μΐ) was added lOx molar excess of NHS-PEG ₄ -N ₃ dissolved in DMSO and the reaction was allowed to proceed for 2 hours at room temperature. To minimize loss of antibody, the reaction was conducted in an Amicon Ultra centrifugal filter unit (30K, 0.5 ml) . Low molecular weight reagents were then removed by Amicon centrifugal filter with repeated addition of PBS - 5 % mannitol - 0.1 % Tween20.

To prepare GCM005-drug conjugate (Scheme 4), 50x molar excess of TGTA and sodium ascorbate in ¾0, lOx molar excess of CUSO ₄ in H20 (final concentration 500 μΜ) and 20x molar excess of N- ( 6-propargyl-D-galactosyl ) -dolastatin 10 in DMSO were added into the solution of GCM005-PEG ₄ -N3. The reactions were performed at room temperature for 45 minutes. The conjugated antibody were purified by Amicon centrifugal filter as above.

Scheme 4. Structure of (N-galactosyl) -Modo-triazole-PEG-GCM005 conj ugate .

EXAMPLE 3. Generation of GCM012 (an anti-EGFRl antibody)

An expression vector with coding sequences for GCM012 heavy- and light chains was constructed to pCHOl .0 backbone. Coding sequence for entire light chain was ordered as a synthetic CHO codon optimized sequence and cloned to pCHOl .0 with AvrII/Bstll07I . Coding sequence for heavy chain was constructed by PCR overlap from two pieces, synthetic sequence for heavy chain variable region and PCR amplified sequence for heavy chain constant region. Full-length PCR product was cloned to the pCHOl.O - GCM012 light chain backbone with EcoRV/PacI. The resulting vector was verified by sequencing and used for transient expression in CHO-S cells.

GCM012 was produced by transfecting CHO-S cells transiently using FreeStyleTM MAX CHO Expression system (Life technologies) according to the manufacturer's instructions. The transfected cell cultures were grown for four days before the culture supernatants were collected by centrifugating at 350 x g for 5 minutes at RT . GCM012 was purified from culture supernatant by Protein A column (High Trap MAb Select Sure; 5 ml; GE Healthcare) and AKTA purifier instrument (GE

Healthcare) as described in purification of GCM023. GCM012 comprises sequences set forth in SEQ ID NOs : 39 and 42. EXAMPLE 4. Generation of GCM014 (GCM012 heavy chain fused to 2G12 scFv variant with additional cysteines)

A vector with CHO optimized coding sequences for GCM014 heavy- and light chain was constructed to a plasmid backbone.

Sequence encoding the C-terminal part (2G12 scFv) of the GCM014 heavy chain was ordered as a synthetic gene, N- terminal part of the GCM014 heavy chain (GCM012) was made as a PCR fragment from the plasmid described in the previous example. Sequences were combined by overlap PCR into EcoRV/PacI site replacing the GCM012 heavy chain and resulting vector was verified by sequencing and used for transient expression in CHO- S cells. The 2G12 scFv part of the GCM014 has two additional cysteines corresponding to G44C in the heavy chain and G100C in the light chain to stabilize the bispecific antibody. Transfecting CHO-S cells for transient GCM014 expression

GCM014 was produced by transfecting CHO-S cells transiently using Freestyle™ MAX CHO Expression system (Life technologies) according to the manufacturer's instructions. The transfected cell cultures were grown for four days before the culture supernatants were collected by centrifugating at 350 x g for 5 minutes at RT .

Glycan analysis of GCM014

N-glycans of GCM014 were analysed from total antibody and Fc fragment. N-glycans were released from SDS denatured GCM014 using PNGase F (ProZyme Inc.) in 20 mM sodium phosphate buffer, pH 7.3, in overnight reaction at +37°C. The released N- glycans were purified with Hypersep C18 and Hypersep Hypercarb (Thermo Scientific) and analysed with MALDI-TOF MS.

In total antibody and Fc fragment the main glycoform was FG0. The MALDI-TOF image of N-glycans released from GCM014 is shown in Figure 1.

For the light chain analysis the domain structure of the antibody was first denatured with guanidine-HCl and the disulphide bonds were reduced with dithiotreitol . Free light chains were then purified with Poros Rl and analyzed by MALDI- TOF. MALDI analysis implicated that sialylated N-glycan SFG2 is the most abundant structure (m/z 26171, Figure 2) . 97% of LCs are glycosylated.

Conjugation of aminooxybutynylacetamide- monomethyldolastatin 10 (ABAA-MODO) to 7-aldehydo-NeuNAc-GCM014

To oxidize the sialic acids in GCM014, the antibody was incubated with 3 mM sodium periodate in 0.1M sodium acetate, pH 4.2. and the 7-aldehydo-NeuNAc-GCM014 thus obtained was purified by ultrafiltration on Amicon centrifugal filter. The outcome of the oxidation reaction was analyzed by liberating the glycosylated light chains and subsequent MALDI-MS analysis as described in Example 5. The mass spectrum showed two major peaks, m/z 26116 and m/z 26351, which were assigned to light chains carrying glycans 7-aldehydo-NeuNAc-G2F and (7-aldehydo- NeuNAc) ₂ ^_ G2F, respectively. Synthesis of 6-azido-6-deoxy-D-galactose

Scheme 5 . Synthesis of 6-azido- 6-deoxy-D-galactose . i) TsCl, pyridine, RT, 22h, 81%; ii) NaN ₃ , DMF, 120°C, 68%; iii) 60% TFA, 50°C, lh, quantitative.

Synthesis of 1 , 2 ; 3 , 4-di-O-isopropylidene- 6-O-tosyl- -D- galactopyranose (Scheme 5, compound 2): 0.39g (1.5mmol) of compound 1 was dissolved in 5ml of dry pyridine under an argon atmosphere. The reaction mixture was cooled on an ice bath and 0.88g (3.1 equiv.) of TsCl was added. The reaction was slowly warmed to RT and stirred overnight. After 22 hours the reaction was diluted with 30ml of CH ₂ CI ₂ and washed with 30ml of ice-cold water. The organic phase was washed with 20ml of 10% (w/v) aqueous CuSC^-solution, 20ml of saturated NaHC03-solution and 20ml H ₂ O . The organic phase was separated, dried over a ₂ S0 ₄ , filtered and concentrated. The crude product was purified by column chromatography (Hexane : EtOAc 1:1) to give compound 2 as a yellowish oil (0.49g, 81%). TLC: R _f = 0.74 (Hexane :EtOAc 1:1). ^X H NMR (600 MHz, CDC1 ₃ , 22°C): δ = 7.81-7.32 (m, 4 H, CH ₃ C ₆ H ₄ SO ₂ ), 5.45 (d, 1 H, Ji , ₂ = 4.9 Hz, H-l), 4.59 (dd, 1 H, J ₃ , 2 = 2.5, J ₃ ,4 = 7.9 Hz, H-3), 4.29 (dd, 1 H, H-2), 4.22- 4.18 (m, 2 H, H-6a, H-4), 4.09 (dd, 1 H, J _6b , ₅ = 6.9, J ₆ b,6a = -10.3 Hz, H-6b), 4.05 (ddd, 1 H, J ₅ , ₄ = 1.9, J ₅ , _6a = 6.2 Hz, H-5) , 2.44 (s, 3 H, CH ₃ C ₆ H ₄ S O2 ) , 1.50, 1.34, 1.31 and 1.28 (each s, each 3 H, 0 ₂ C(CH ₃ ) ₂ ) ppm.

Synthesis of 1 , 2 ; 3 , 4-di-O-isopropylidene- 6-deoxy- 6- azido-a-D-galactopyranose (Scheme 5, compound 3) . To a solution containing 1.5g (3.7mmol) of compound 2 in 20ml dry DMF (under an argon atmosphere) was added 1.7g (7 equiv.) NaN ₃ and the resulting mixture was stirred at 120°C overnight. After 18 hours, the reaction mixture was brought to RT, diluted with 20ml CHCI3, filtered and concentrated. The crude product was purified by column chromatography (Hexane : EtOAc 3:1) to give compound 3 as a colorless oil (0.7g, 68%). TLC: R _f = 0.52 (Hexane : EtOAc 3:1). ¹ NMR (600 MHz, CDC1 ₃ , 22°C): δ = 5.55 (d, 1 H, Ji , ₂ = 5.1 Hz, H-l), 4.63 (dd, 1 H, J ₃ , ₂ = 2.5, J ₃ , ₄ = 8.1 Hz, H-3), 4.33 (dd, 1H, H-2), 4.19 (dd, 1 H, J ₄ , ₅ = 2.0 Hz, H-4), 3.92 (ddd, 1 H, J ₅ , ₆ b = 5.3, J ₅ ,6a = 7.8 Hz, H-5), 3.51 (dd, 1 H, J _6a ,6b = -12.9 Hz, H- 6a), 3.36 (dd, 1 H, H-6b) , 1.55, 1.46, 1.35 and 1.34 (each s, each 3 H, 0 ₂ C(CH ₃ ) ₂ ) ppm.

Synthesis of 6-azido- 6-deoxy-D-galactose (Scheme 5, compound 4) . 80mg (0.3mmol) of compound 3 was dissolved in 3ml 60% TFA and the resulting mixture was stirred at 50°C for 1 hour. The mixture was then diluted with water and concentrated to give 6-azido- 6-deoxy-D-galactose as a colorless oil (60mg, quantitative, furanose : pyranose 3:97, alpha _P y _ran ose : beta _P y _ran ose 35:65). Selected NMR-data: ¹ NMR (600 MHz, D ₂ 0, 22°C): δ = 5.28 (d, 1 H, Ji , 2 = 4.7 Hz, H-l _furanose ) , 5.26 (d, 1 H , Ji , ₂ = 3.9 Hz, H- lOipyranose) , 5.22 (d, 1 H, Ji , ₂ = 3.4 Hz, H"lfuranose ), 4.60 (d, 1 H,

Ji , 2 ⁼ 7.8 Hz, Η-1β _{ργΓ¾η03θ} ) .

Synthesis of N- (6-azido-6-deoxy-D-galactosyl) -dolastatin 10

Sodium cyanoborohydride (160ymol) and 6-azido- 6-deoxy- D-galactose (95ymol) were added to the solution of monomethyldolastatin 10 (MODO) (2.5ymol) in DMSO (0.6ml). The mixture was stirred at 60 °C for three days. The title compound (N- ( 6-N ₃ -Gal ) -MODO) was isolated by reversed-phase chromatography on a column of Gemini-NX-5u C-18 (Phenomenex) . Dolastatin derivative carrying an aminooxy-functionality

2- [N- (tert-butoxycarbonyl) aminooxy] -N- (butynyl) acetamide (Boc-ABAA) was prepared as follows: Boc-aminooxyacetic acid (Novabiochem) (0.41 g, 2.1 mmol) was dissolved in 7 ml dry THF (under argon atmosphere) and the mixture was cooled on an ice bath. 0.24 ml (2.1 mmol, 1 equiv.) NMM and 0.28 ml (2.1 mmol, 1 equiv.) IBCF were added and the reaction mixture was stirred for 0.5 h at 0 °C . 0.18 ml (2.1 mmol, 1 equiv.) of 1- amino-3-butyne was added and the resulting mixture was brought to RT and stirred for an additional 1.5 h. The mixture was then filtered and concentrated and the crude product was dissolved in 20 ml Et ₂ 0 and washed with 10 ml 0.1 M NaOH, 10 ml 1 M HC1 and 10 ml brine. The organic phase was dried with a ₂ S0 ₄ , filtered and concentrated. The crude product was purified by column chromatography (hexane : EtOAc 1:2) to give the title compound as a white solid. TLC : R _f = 0.34 (in hexane:EtOAc 1:2). Gal ^X H NMR (600 MHz, CDC13, 22 °C) : δ 8.25 (br s, 1 H, NH) , 7.48 (s, 1 H, NH) , 4.33 (s, 2 H, OC¾CO) , 3.49 (ap q, 2 H, J = 6.8 Hz, NHC¾CH ₂ C≡CH) , 2.44 (ap td, 2 H, J = 2.6, 6.8 Hz, NHCH ₂ C¾C≡CH) , 1.99 (ap t, 1 H, J = 2.6 Hz, NHCH ₂ CH ₂ C≡CH) and 1.49 (s, 9 H, OC(CH ₃ ) ₃ ) ppm.

Boc-ABAA was conjugated to N- ( 6-N ₃ -Gal ) -MODO by copper (I) catalyzed azide-alkyne cycloaddition reaction: The reaction contained 2.5 ymol N- ( 6-N ₃ -Gal ) -MODO, 6,3 ymol Boc-ABAA (2,5 x molar excess to N- ( 6-N ₃ -Gal ) -MODO) , 25 ymol Na-ascorbate (10 x molar excess to N- ( 6-N ₃ -Gal ) -MODO) and 5 ymol of CuS0 ₄ (2 x molar excess to N- ( 6-N ₃ -Gal ) -MODO) . Boc-ABAA and N- ( 6-N ₃ -Gal) - MODO were dissolved in DMSO and Na-ascorbate and CuS0 ₄ in MilliQ- H ₂ 0 before adding to the reaction. Total volume of the reaction was 117 μΐ containing 64% DMSO. Reaction was carried out for 1.5 hours at RT . The conjugation was stopped with 40 μΐ of 0,5M EDTA pH 8 (20 μιηοΐ EDTA) . Progress of the reaction was analyzed with MALDI-TOF MS using 2 , 5-dihydroxybenzoic acid matrix in the positive ion reflector mode. Major signal was observed at m/ z 1224.6, which corresponds to [M+Na] ⁺ ion of the expected click- reaction product Modo-Boc- aminooxybutynylacetamide (Boc-

ABAA-MODO) .

Boc-ABAA-MODO was purified by solid-phase extraction on Bond-Elut C18 extraction cartridge. To obtain unprotected aminooxy drug, ABAA-MODO, the Boc-protecting group was removed by incubating in dichloromethane-TFA (12.5:1), and the product MODO-ABAA was isolated by reversed-phase chromatography using Gemini NX C18 column (Phenomenex) using a acetonitrile gradient in 20 mM ammonium acetate, pH 5.6.

Conjugation of ABAA-MODO to 7-aldehydo-NeuNAc-GCM014

ABAA-MODO was conjugated to 7-aldehydo-NeuNAc-GCM014 in an oxime ligation reaction by incubating 500 yg of antibody with 34 nmol of ABAA-MODO in 0.08M sodium acetate, pH 4.2. The conjugate (Scheme 6) was purified by ultrafiltration and light chains were isolated as above to analyze the reaction outcome. MS analysis showed a major signal at m/z 28271, which was assigned to (7- aldehydo-NeuNAc) 2-G2F-LC carrying two oxime linked ABAA-MODO units .

Scheme 6. Structure of MODO-ABAA-GCM014. For clarity, only sialic acid and galactose residues of the N-glycan are shown.

EXAMPLE 5. Generation of GCM023 (GCM012 heavy chain fused to 2G12 scFv variant [additional cysteines in scFv and I19N and F78S in heavy chain] )

I506N and F565S mutations (corresponding to I19N and

F78S in 2G12 heavy chain) were made by overlapping PCR, amplifying Ascl-Pacl fragment of GCM012 heavy chain as three overlapping parts, and assembling the final plasmid comprising also GCM012 light chain sequence.

IRES2-eGFP fragment was added to the construct to aid the identification of high-expressing clones. IRES2-eGFP sequence was cut from helper plasmid as Eco32I-fragment and cloned into Bstll07I-site right after light chain.

Transfecting CHO-S cells for stable GCM023 expression using Freestyle MAX reagent

GCM023 (cloned in an IRES-eGFP vector) was transfected into CHO-S cells to develop stable cell pools for GCM023 production using Freedom CHO-S kit (Life Technologies) according to the manufacturer's instructions. Negative controls (no Freestyle MAX reagent, no DNA) as well as the expression vector (pCHOl.O) alone were included. GCM023 plasmid and the expression vector were linearized using Ndel prior to transfection to improve the transfection efficiency. Two transfections with the GCM023 plasmid were performed. Selecting stable transfectants for GCM023 expression

Selection phase 1

Selection phase 1 was conducted after transfection according to the manufacturer's instructions using selection reagents puromycin (Invitrogen) and methotrexate (Sigma) to select the stable transfectants . For each transfection two flasks (125 ml Erlenmeyer flask, Corning) were seeded having different selection reagent concentrations. Negative controls (no Freestyle MAX reagent, no DNA) were discarded on day 14 of selection phase 1 (almost no live cells) . Selection phase 1 was completed when the viability of the samples exceeded 85% and pools of each sample were cryopreserved .

Selection phase 2

In the beginning of selection phase 2 each sample flask was further divided into two having increased selection reagent concentrations according to the Freedom CHO-S Kit manual. Selection was complete when the viability exceeded 90 % and pools of each sample were again cryopreserved. Analysing the selections phase 2 cell pools for eGFP expression using flow cytometry

At the end of selection phase 2 the cell pools were analysed for eGFP expression, which correlated with the level of GCM023 produced. The eGFP expression of the cell pools was analysed by flow cytometry (FACSAria II, BD Biosciences) and the data analysis was done by FACSDiva software. About 1 x 10 ^s cells were taken from each sample, the cells were centrifuged at 200 x g for 5 minutes and the cells were suspended in 0.5 ml Complete CD FortiCHO™ medium (Life technologies) . The cells were filtered through cell strainer caps (35 ym nylon mesh, BD Falcon) and 10 000 events were recorded from each sample. The sample having only the expression vector transfected was used as a negative control in the analysis. The amount of eGFP-positive cells was about 3-100 % in the samples.

Sorting

Based on the flow cytometry analysis of selection phase 2 cell pools, a sample (99.9 % eGFP - positive) was chosen to be used in sorting. The most eGFP -positive cells (15.5~6 ) were sorted using 2-way purity method aiming to get a cell pool with enhanced GCM023 producing capability. The sorted cells were collected into 15 ml Falcon tube having 0.5 ml Complete CD FortiCHO™ medium. About 6.5 x 10 ^s cells were sorted in total. The sorted cells were centrifuged (300 x g, 5 min, RT) and suspended in 20 ml Complete CD FortiCHO™ medium including the selection reagents (50 yg/ml Puromycin; 1000 nM methotrexate) and penicillin/streptomycin. Culture of GCM023 producing cells

Sorted cells or the high expressing cell pool were grown for six days in Complete CD FortiCHO™ medium, 0.3 x 10 ^s cells/ml; V=30 ml. On day five a 3 ml of CHO CD Efficient Feed B (Life technologies) was added to each flask (125 ml Erlenmeyer flask, Corning) . The culture medium was collected at day 6 by centrifugating at 350 x g, 5 min, RT .

Purification of GCM023 from culture supernatant using Protein A column The culture medium was filtered (HPF Millex HV PVDF filter; 0.45 ym) and NaCl was added to the medium to a final concentration of 150 mM. The GCM023 antibody was purified using Protein A column (High Trap MAb Select Sure; 5 ml; GE Healthcare) and AKTA purifier instrument (GE Healthcare) . The column was equilibrated with 20 mM Na-phosphate - 150 mM NaCl, pH 7.3 before sample application. The unbound material was washed with 50 mM Tris - 250 mM arginine-HCl - 1M NaCl, pH 9 and with 20 mM Na-phosphate - 150 mM NaCl, pH 7.3. GCM023 was eluted with 0.1M Na-citrate, pH 3. GCM023 containing fractions were collected, neutralized with 2M Tris-HCl, pH 9 and pooled. The pooled fractions were concentrated using Amicon Ultra-4 Centrifugal filter unit, 30K (Millipore) . The GCM023 concentration was determined by using NanoDrop 1000 (Thermo Scientific) .

SDS-PAGE

GCM023 produced from high expressing cell pool was ran in SDS-PAGE (4-15 % Mini-Protean TGX gel; BioRad) , in the presence of reducing (Figure 3, lane 1) and non-reducing (Figure 3, lane 2) sample buffer. The size of the light chain is about 37 kDa and that of heavy chain about 100 kDa . The non-reduced protein moves around the place of the 260 kDa molecular marker.

N-glycans were released from GCM023 and purified as described above. The main glycoform in the GCM023 heavy chain is FG0 (m/z 1485, Figure 4) .

The N-glycosylation of light chains was analysed as described above. MALDI analysis implied sialylated N-glycans in the light chains SFG2 being the most abundant structure (Figure 5) . 94% of LCs were glycosylated.

30 yg of antibody was digested with 30 U of FabRICATOR (Genovis) , +37°C, 60 min, producing F(ab')2 fragment and two Fc- scFv fragments. Digested samples were purified using Poros Rl filter plate (Glyken corp.) and the Fc-scFv fragments were analysed using MALDI-TOF MS. According to the analysis, at least over 94% of the scFv glycosylation site (2G12, HC:I19N) in the Fc-scFv fragment was glycosylated (Figure 6) .

Conjugating DMl-DBCO to GCM023 Galactosylation and 9-azido- sialylation of GCM023

Terminal galactose-residues in GCM023 N-glycans were sialylated with a-2, 6 sialyltransferase : 100 μΜ antibody, 30 mM CMP-NeuNAc and 0,05 yg/μΐ ST6Gal-l (hum rec, Roche Diagnostics) in 50 mM MOPS pH 7,2 were incubated o/n at +37°C. To analyze the reaction progress, Fc-ScFv-fragments were released from a small aliquot of the reaction mixture with Fabricator enzyme and recovered using reversed-phase extraction on hand ^¬ made Poros Rl tips. Light chains were released by treatment with 6M guanidine-HCl and dithiothreitol , and recovered using Poros Rl tips. MALDI-TOF MS analysis of purified Fc-ScFv revealed major signal at m/z 27210, which was assigned to doubly-charged (z=2) ion of Fc-ScFv corresponding to GOF in Fc-N-glycans and SGIF in ScFv. Another signal at m/z 27438 (z=2) was revealed corresponding to GOF in Fc-N-glycans and S2G2F in ScFv. A minor signal at m/z 27291 (z=2) was also detected corresponding to GOF in Fc-N-glycans and SG2F in ScFv. MALDI-TOF MS analysis of purified LC revealed major signal at m/z 26466 corresponding to LC with S2G2F glycan. Minor signals at 26012 and 26177 corresponded to LC with SGIF and SG2F.

Sialylated GCM023 was purified with HiTrap Protein G column (GE Healthcare) using 0.02 M Na-phosphate pH 7.0 as the binding buffer and 0.1 M citric acid pH 2.6 as the elution buffer. Purified antibody was subjected to galactosylation and sialylation using UDP-galactose and CMP- 9-azido-N-acetyl- neuraminic acid as the donor substrates. β-1,4- galactosyltransferase- and a-2 , 6-sialyltransferase-reactions were done in succession without removal of the galactosylation components. In short, 100 μΜ antibody, 20 mM UDP-Gal, 4 mU/μΙ β- 1,4-GalT (bovine milk, Sigma), 10 mM MnCl ₂ in 50 mM MOPS pH 7,2 were incubated o/n, after which CMP- 9-azido-NeuNAc and ST6Gall (hum rec, Roche Diagnostics) were added to final concentrations of 15 mM and 0,05 μg/μl. Incubation was continued o/n at +37°C. Fc-ScFv-fragments were released from small aliquot of reaction mixture with Fabricator enzyme and recovered using Poros Rl tips. Light chains were released from a small aliquot by treatment with 6M guanidine-HCl and dithiothreitol, and recovered using Poros Rl tips. MALDI-TOF MS analysis of purified Fc-ScFv revealed major signal at m/z 27761, which was assigned to doubly-charged ion of Fc- ScFv carrying 9-azido-NeuNAc- G2F in Fc-N-glycans + S2G2F in ScFv, and Fc-ScFv carrying 9- azido-NeuNAc-G2F in Fc-N-glycans and 9-azido-NeuNAc-SG2F in ScFV. The galactosylated/ 9-azido-sialylated antibody was purified with Protein G as above.

Synthesis of DMl-DBCO

3.9 ymol DM1, 2.5 molar excess of iodoacetic acid in DMF (33 μΐ), 67 μΐ DMF and 90 μΐ 200 mM NH ₄ HCO ₃ were stirred at RT for one hour. The crude reaction mixture was analysed by MALDI-TOF mass spectra using 2 , 5-dihydroxybenzoic acid matrix, showing expected mass for DMl-S-CH ₂ COOH (m/z 818 [M+Na] ⁺ ). The crude DMl-S-CH ₂ COOH, 3.5 molar excess of DBCO-NH ₂ (Sigma) in DMF (200 μΐ) and 26 mg (95 μιηοΐ) DMT-MM in DMF (500 μΐ) were stirred at RT for overnight. The crude reaction mixture was analysed by MALDI-TOF mass spectra using 2 , 5-dihydroxybenzoic acid matrix, showing expected mass for DMl-DBCO (m/z 1076 [M+Na] ⁺ ). DMl-DBCO was purified by reversed-phase chromatography on a column of Gemini-NX-5u C-18 (Phenomenex) .

Conjugation of DMl-DBCO and azido-sialylated GCM023

DMl-DBCO was conjugated to galactosylated and 9-azido- sialylated GCM023 as follows: 20 μΜ antibody in 4 "6 mannitol , 0,1 % Tween, PBS was incubated with 400 μΜ DMl-DBCO in the presence of 20% propylene glycol and 6% DMSO. Reaction was allowed to proceed 16 h at room temperature after which unbound DMl-DBCO was removed by repeated additions of 5 % mannitol, 0,1 % Tween, PBS and centrifugations through Amicon Ultracel 30 k centrifugal filter. DMl-DBCO-linked antibody (Scheme 7) was recovered from the concentrator and a small sample was taken for Fc-analysis, which revealed major signals at m/z 28275 and 28815. The signal m/z 28275 was assigned to DMl-DBCO-9-azido- NeuNAc-G2F in Fc N-glycans and S2G2F in ScFv N-glycans. m/z 28815 was assigned to DMl-DBCO-9-azido-NeuNAc-G2F in Fc N- glycans and DMl-DBCO-9-azido-NeuNAc-SG2F in ScFv N-glycans.

Scheme 7. Structure DMl-DBCO-9-azido-NeuNAc-GCM023. For clarity, only sialic acid and galactose residues of the N-glycan are shown.

Considering that light chain was free of 9-azido-sialic acids, and thus unreactive with DM1-DBCO, the antibody-drug- conjugate was thus a mixture of antibodies having either 2 (DAR2) or 4 (DAR4) attached drugs.

Synthesis of DM1-DBCO-GCM023 conjugate

GCM023-PEG4-N3 was prepared as described in Example 2. To prepare GCM023-DM1 conjugate (Scheme 8), lOx molar excess per azido group of DM1-DBCO (Example 5) in DMSO was added into the solution of GCMO23-PEG ₄ -N3. The reaction was performed at room temperature for 2 hours. The conjugated antibody was purified by Amicon centrifugal filter as above. To analyze the reaction progress, light chains were isolated as described in Example 5. The analysis revealed a major signal at m/z 27500, corresponding to light chain carrying one DM1-DBCO unit.

Scheme 8. Structure of DM1-DBCO-GCM023 conjugate. Synthesis of DBCO-Val-Cit-PAB-MODO-GCM023 conjugate

Synthesis of Val-Cit-PAB-MODO

6.5 mg (8 ymol) monomethyldolastatin (Modo) in DMF (200 μΐ) , 2 molar excess of Fmoc-Val-Cit-PAB-pnp (Concortis Biosystems) , 0.3 mg (2 ymol) HOBt in DMF (28 μΐ), 7 μΐ (40 μιηοΐ) diisopropylethylamine and 65 μΐ DMF were stirred for two days at room temperature. The crude reaction mixture was analysed by MALDI-TOF mass spectra using 2 , 5-dihydroxybenzoic acid matrix, showing expected mass for Fmoc-Val-Cit-PAB-MODO (m/z 1420 [M+Na] ⁺ ) .

Fmoc removal was carried out by adding 150 μΐ of diethylamine and by stirring at room temperature overnight. MALDI-TOF mass analysis using 2 , 5-dihydroxybenzoic acid matrix showed the generation of expected deprotected product (m/z 1198 [M+Na] ⁺ ) .

Val-Cit-PAB-MODO was purified by Akta purifier (GE

Healthcare) HPLC instrument with Gemini 5 μιη NX-C18 reverse phase column (21.1 x 250 mm, 110 A, AXIA (Phenomenex) ) eluted with ACN gradient in aqueous ammonium acetate. Synthesis of DBCO-Val-Cit-PAB-MODO

~2 μιηοΐ Val-Cit-PAB-MODO, ~5 molar excess of DBCO-NHS ester in DMF (126 μΐ) and 3.5 μΐ diisopropylethylamine were stirred at RT for three hours. The crude reaction mixture was analysed by MALDI-TOF mass spectra using 2 , 5-dihydroxybenzoic acid matrix, showing expected mass for MODO-Val-Cit-PAB-DBCO (m/z 1485 [M+Na] ⁺ ) .

MODO-Val-Cit-PAB-DBCO was purified by reversed-phase chromatography as described above.

Conjugation of DBCO-Val-Cit-PAB-MODO with GCM023-PEG4-N ₃

GCM023-PEG4-N3 was prepared as described in Example 2. To prepare GCM023-drug conjugate (Scheme 9) , lOx molar excess per azido group of DBCO-Val-Cit-PAB-MODO in DMSO was added into the solution of GCMO23-PEG ₄ -N3. The reaction was performed at room temperature for 2 hours. The conjugated antibody was purified by Amicon centrifugal filter as above.

To analyze the reaction progress, light chains were isolated as described in Example 5. The analysis revealed a major signal at m/z 27906, corresponding to light chain carrying one DBCO-Val-Cit-PAB-MODO unit.

Scheme 9. Structure of GCM023-DBCO-Val-Cit-PAB-MODO conjugate.

Synthesis of MODO- REA-DBCO-GCMO23 conjugate

Synthesis of MODO-TREA (1- [MODO-Gal] -1 , 2, 3-triazol-4-ethylamine)

12 ymol N- ( 6-N ₃ -Gal ) -MODO (Example 4) in DMSO (40 μΐ) , 2x molar excess of l-amino-3-butyne in DMSO (20 μΐ) , 3.1 mg (19 mmol) CuS0 ₄ in MQ (50 μΐ) , 19.2 mg Na-ascorbate in MQ (50 μΐ) , 90 μΐ DMSO and 400 μΐ MQ were stirred at RT for 2.5 hours. The crude reaction mixture was analysed by MALDI-TOF mass spectra using 2 , 5-dihydroxybenzoic acid matrix, showing expected mass for MODO-TREA (m/z 1051 [M+Na]).

MODO-TREA was purified by Akta purifier (GE Healthcare) HPLC instrument with Gemini 5 μιη NX-AXIA-C18 reversed phase column (21.2 x 250 mm, 110 A (Phenomenex) ) eluted with ACN gradient in aqueous ammonium acetate.

Synthesis of MODO-TREA-DBCO

8 ymol MODO-TREA, 5x molar excess of DBCO-NHS ester

(Jena Bioscience) in DMF (1 ml) and 16 μΐ diisopropylethylamine were stirred at RT for three hours. The crude reaction mixture was analysed by MALDI-TOF mass spectra using 2,5- dihydroxybenzoic acid matrix, showing expected mass for MODO- TREA-DBCO (m/z 1338 [M+Na] ) .

MODO-TREA-DBCO was purified by Akta purifier (GE Healthcare) HPLC instrument with Gemini 5 ym NX-AXIA-C18 reversed phase column (21.2 x 250 mm, 110 A (Phenomenex)) eluted with ACN gradient in aqueous ammonium acetate.

Conjugation of MODO-TREA-DBCO with GCM023-PEG4-N ₃

GCM023-PEG4-N ₃ was prepared as described in Example 2.

To prepare GCM023-MODO-TREA-DBCO (Scheme 10), lOx molar excess per azido group of MODO-TREA-DBCO in DMSO was added into the solution of GCMO23-PEG ₄ -N ₃ . The reaction was performed at room temperature for 2 hours. The conjugated antibody was purified by

Amicon centrifugal filter as above.

To analyze the reaction progress, light chains were isolated as described in Example 5. The analysis revealed a major signal at m/z 27770, corresponding to light chain carrying one MODO-TREA-DBCO unit.

Scheme 10. Structure of MODO-TREA-DBCO-GCM023 conjugate.

EXAMPLE 6. Conjugation of aminooxy functionalized MMAF to 7- aldehydo-NeuAc-cetuximab

10 mg monomethylauristatin F (Concortis) was dissolved in acetonitrile (2.5 ml). lOx molar excess of Boc-aminooxyacetic acid (Novabiochem) and DMT-MM were added. 25 μΐ of diisopropylethylamine was added and the reaction mixtures were stirred overnight at room temperature. MALDI-TOF MS analysis showed the formation of the expected product, monomethylauristatin-boc-aminooxyacetic acid amide, (MMAF-ABAA) m/z=927 [M+Na] . Boc-protecting group was removed as described in Example 4, and the deprotected product was purified by reversed- phase chromatography on a column of Gemini-NX-5u C-18.

7-aldehydo-NeuAc-cetuximab was prepared as described in Example 4, and was conjugated with 300x molar excess of MMAF- AOAA in 0.1 M sodium acetate buffer pH 5.5.

The Fc-fragment of MMAF-AOAA-Cetuximab conjugate (Scheme 11) was isolated as described in Example 5 and analyzed by MALDI-TOF MS. The spectrum of Fc-fragment showed a major signal at m/z 26614, corresponding to MMAF-AOAA-Cetuximab conj ugate .

Scheme 11. Structure of MMAF-AOAA-Cetuximab conjugate. For clarity, only sialic acid and galactose residues are shown.

EXAMPLE 7. Antibody binding to cells and internalization

Cells were suspended in 0.1% BSA-PBS-buffer at 5-10 x

10 ^s cells/ml concentration and 100 μΐ was used for staining. Primary antibody was used at 20 μg/ml and secondary antibody when needed at 2 μg/ml concentration and staining was carried out for 30 minutes on ice. Cells were analysed with LSRII flow cytometer (Becton Dickinson) and FACSDiva 6.2 software (see Table 1) For 2G12 and GCM005 antibody analysis HEK-cells were cultured in the presence of kifunensine (a mannosidase inhibitor) . After 3 days culture with kifunensine (Cayman Chemical) at 12 μg/ml concentration high-mannose glycans appear on the cell surface. Cetuximab and GCM012 were used as a control.

Table 1. Percentage of stained cells.

Internalization assays FACS analysis

HSC-2, FaDu, Skov-3 and HEK cells (2xl0 ⁵ ) were seeded on a 24-well plate and allowed to grow for 24h. Thereafter the cells were incubated 3h either at +37°C or at +4°C in 300μ1 media containing 2 yg/ml AlexaFluor488 labeled 2G12, cetuximab, or GCM005. The cells were washed two times with PBS, detached by incubating with ΙΟΟμΙ Trypsin-EDTA, lOmin at +37°C, neutralized with 300 μΐ media, resuspended in PBS, and analyzed in a flow cytometer (FACS LRS II) . The mean fluorescence intensity of each samples were calculated by the software (FACS Diva) . Table 2 shows the results of the internalization assay (measured as mean of all fluorescent events) . Table 3 shows an "internalization efficiency" measured as a fold change of fluorescent intensity to +4C value (= ratio +37C/+4C) . Table 4 shows fold increase of internalization of the bispecific antibody GCM005 compared to its components 2G12 or cetuximab.

Table 2. Mean fluorescent intensity of antibodies at +4°C (cell surface bound) and +37°C (internalized) .

HEK HEK Fadu Fadu HSC2 HSC2 SKOV- SKOV-3 3

( +4C) (+37C) ( +4C) (+37C) ( +4C) (+37C) ( +4C) (+37C)

2G12 82 414 127 887 387 3457 236 867 cetuximab 170 158 3877 6906 49990 82296 1810 2746

GCM005 215 1551 4246 12228 54667 107697 1849 5255

Table 3. Fold change of an antibody internalization as a ratio of +37°C to +4°C.

Table 4. Fold increase of GCM005 compared to its "components 2G12 or cetuximab at +37°C.

Microscope analysis

HSC-2 cells (5xl0 ⁴ ) were seeded on a chamber slides and allowed to grow for 24h. Thereafter the cells were incubated 3h either at +37°C or at +4°C in ΙΟΟμΙ media containing 5 yg/ml AlexaFluor488 labeled GCM005. The cells were washed two times with PBS and fixed with 4% paraformaldehyde for 20 min. The cells were mounted (Prolong Gold antifade reagent with DAPI) and photographed with fluorescence microscopy (Zeiss Axio Scope Al) . GCM005 binds to cell surface at 4°C but after incubation at 37°C, antibody was internalized and most staining was seen inside the cells (Figure 7) .

In vitro cell assay

Toxicity of antibody drug conjugates (ADC) was tested by in vitro cell assay. Appropriate amount of cells was plated the day before on 96-well plate. ADCs were diluted in cell culture medium and sterile filtered with a syringe and a 0.22 μιη filter. Logarithmic dilution series from ADC was prepared and applied to cells in triplicate. Cells with ADC were cultured for three to four days at +37°C and 5% CO ₂ . Cell proliferation and viability was evaluated using PrestoBlue Cell Viability reagent (Life Technologies) according to the manufacturer's protocol. Cell culture medium was used as a background control. Viability of ADC treated cells was compared to untreated (normal) cells (= percentage of living cells) (see Tables 5-7) . Table 5.

Table 6.

Table 7.

HEKa Percentage of untreated

ADC 1 pM 10 pM 100 1 nM 10 nM 100

pM nM

2G12-N ₃ NeuNAc-DMl- 88, 9 91, 4 87,1 22,2 18,8 19, 3

DBCO GCM023-N ₃ NeuNAc- 104,2 88, 6 92, 0 34, 7 29, 1 34, 0

DM1-DBCO

As is clear for a person skilled in the art, the invention is not limited to the examples and embodiments described above, but the embodiments can freely vary within the scope of the claims.