HK1191343A

HK1191343A - Monovalent antigen binding proteins

Info

Publication number: HK1191343A
Application number: HK14104470.5A
Authority: HK
Inventors: Birgit Bossenmaier; Hubert Kettenberger; Christian Klein; Klaus-Peter KEUNKELE; Joerg Thomas Regula; Wolfgang Schaefer; Manfred Schwaiger; Claudio Sustmann
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2011-02-28
Filing date: 2012-02-24
Publication date: 2014-07-25

Description

Monovalent antigen binding proteins

The present invention relates to monovalent antigen binding proteins with a CH1-CL domain exchange, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Background

A variety of mono-or multispecific, monovalent or multivalent engineered antibody derivatives have been developed and evaluated in recent 20 years (see, e.g., Holliger, P., et al, Nature Biotech23(2005) 1126-1136; Fischer, N., and Leger, O., Pathiology 74(2007) 3-14).

For certain antigens, such as c-Met, monovalent antibodies have different properties compared to their corresponding bivalent form, e.g. lack of anti-function or reduced receptor internalization upon antibody binding, and thus represent an advantageous form for therapeutic use. For example, WO 2005/063816 relates to monovalent antibody fragments as therapeutic agents.

US 2004/0033561 describes a method of generating monovalent antibodies based on co-expressing a VH-CH1-CH2-CH3 antibody chain with a VL-CL-CH2-CH3 antibody chain; however, a disadvantage of this approach is the formation of a binding inactive homodimer of the VL-CL-CH2-CH3 chain, as depicted in FIG. 2. Due to similar molecular weights, it is difficult to separate such homodimeric by-products.

WO 2007/048037 also relates to monovalent antibodies based on co-expressing VH-CH1-CH2-CH3 antibody chains with VL-CL-CH2-CH3 antibody chains, but with a tagging moiety attached to the heavy chain for easy purification of heterodimers from difficult to separate homodimeric by-products.

WO 2009/089004 describes another possibility to generate heterodimeric monovalent antibodies using electrostatic steering effects.

WO 2010/145792 relates to tetravalent bispecific antibodies, wherein a reduction of mismatch by-products of similar weight results in higher yields of the desired bispecific antibody.

Summary of The Invention

The invention encompasses monovalent antigen binding proteins comprising

a) A modified heavy chain of an antibody that specifically binds an antigen, wherein a VH domain is replaced with a VL domain of the antibody; and

b) a modified heavy chain of the antibody, wherein the CH1 domain is replaced by the CL domain of the antibody.

In one embodiment of the invention, the monovalent antigen binding protein according to the invention is characterized in that

a) The CH3 domain of the modified heavy chain of the antibody of (a) and the CH3 domain of the modified heavy chain of the antibody of (b) are each in contact at an interface comprising the original interface between the CH3 domains of the antibody;

wherein the interface is altered to facilitate formation of a monovalent antigen binding protein, wherein the alteration is characterized by

i) The CH3 domain of one heavy chain is altered,

such that, in the original interface of the CH3 domain of one heavy chain that contacts the original interface of the CH3 domain of another heavy chain within a monovalent antigen binding protein,

replacement of amino acid residues with amino acid residues having a larger side chain volume, thereby creating a bulge in the interface of the CH3 domain of one heavy chain that can be placed in a cavity in the interface of the CH3 domain of the other heavy chain

And

ii) altering the CH3 domain of the other heavy chain,

such that, in the original interface of the second CH3 domain in contact with the original interface of the first CH3 domain within the monovalent antigen binding protein,

the amino acid residue was replaced with an amino acid residue having a smaller side chain volume, creating a cavity within the interface of the second CH3 domain in which a bulge within the interface of the first CH3 domain could be placed.

The amino acid residue with larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), and the amino acid residue with smaller side chain volume is selected from alanine (A), serine (S), threonine (T), valine (V).

In one embodiment of the invention, the monovalent antigen binding protein according to the invention is further characterized in that

The two CH3 domains were further altered by introducing cysteine (C) as an amino acid at the corresponding position of each CH3 domain, enabling the formation of a disulfide bridge between the two CH3 domains.

In one embodiment, the monovalent antigen binding protein according to the invention is characterized in that it is of the human IgG isotype.

In one embodiment, a monovalent antigen binding protein according to the invention is characterized by comprising

a) A modified heavy chain comprising SEQ ID NO: 1; and

b) a modified heavy chain comprising SEQ ID NO: 2;

or

a) A modified heavy chain comprising SEQ ID NO: 3; and

b) a modified heavy chain comprising SEQ ID NO: 4;

or

a) A modified heavy chain comprising SEQ ID NO: 5; and

b) a modified heavy chain comprising SEQ ID NO: 6;

or

a) A modified heavy chain comprising SEQ ID NO: 7; and

b) a modified heavy chain comprising SEQ ID NO: 8;

or

a) A modified heavy chain comprising SEQ ID NO: 9; and

b) a modified heavy chain comprising SEQ ID NO: 10;

or

a) A modified heavy chain comprising SEQ ID NO: 11; and

b) a modified heavy chain comprising SEQ ID NO: 12.

In one aspect of the invention, the monovalent antigen binding protein according to the invention is characterized in that the modified heavy chains of a) and b) are of IgG1 isotype and the antigen binding protein is afucosylated, with an amount of fucose of 80% or less (preferably 65% to 5%) of the total amount of oligosaccharides (sugars) at Asn 297.

The invention also encompasses a method of making a monovalent antigen binding protein according to the invention,

which comprises the following steps

a) Transformation of a host cell with a vector comprising a nucleic acid molecule encoding a monovalent antigen binding protein according to the invention

b) Culturing the host cell under conditions that allow synthesis of the monovalent antigen binding protein molecule; and

c) recovering the monovalent antigen binding protein molecule from the culture.

The invention also encompasses nucleic acids encoding monovalent antigen binding proteins according to the invention.

The invention also encompasses a vector comprising a nucleic acid encoding a monovalent antigen binding protein according to the invention.

The invention also encompasses a host cell comprising said vector.

The invention also encompasses compositions, preferably pharmaceutical or diagnostic compositions, of monovalent antigen binding proteins according to the invention.

The invention also encompasses a pharmaceutical composition comprising a monovalent antigen binding protein according to the invention and at least one pharmaceutically acceptable excipient.

The invention also comprises a method of treating a patient in need of therapy, said method being characterized by administering to the patient a therapeutically effective amount of a monovalent antigen binding protein according to the invention.

The antigen binding proteins according to the present invention are based on the principle that the chains VL-CH1-CH2-CH3 and VH-CL-CH2-CH3 form only heterodimers, and do not form homodimer by-products of similar structure and molecular weight that are difficult to separate. The effect of this modification is not only mainly a reduction of side products, but also the only side products formed change from homodimeric side products with similar size to high molecular weight tetramers (fig. 1D). This high molecular weight tetramer can then be easily removed by SEC or other molecular weight separation techniques.

Due to the difference in molecular weight (approximately doubled) and structure, the dimer by-product formed can be easily separated (fig. 1D). Thus, purification can be achieved without the need to introduce additional modifications (e.g., genetically introduced tags).

It has also been found that the monovalent antigen binding proteins according to the invention have valuable characteristics, such as biological or pharmacological activity (e.g. ADCC, or antagonism of biological activity and lack of antagonistic activity). It can be used, for example, for the treatment of diseases, such as cancer. Monovalent antigen binding proteins also have highly valuable pharmacokinetic properties (e.g., half-life (term t 1/2) or AUC).

Description of the drawings

FIG. 1A) schematic representation of a monovalent antigen binding protein according to the invention with CH1-CL domain exchange (abbreviated MoAb) based on VL-CH1-CH2-CH3 and VH-CL-CH2-CH3 chains. B) Schematic representation of a MoAb according to the invention comprising a knob-in-hole in the CH3 domain. C) Schematic representation of dimeric monovalent antigen binding proteins (MoAb dimers formed as by-products, which can be easily separated due to differences in structure and molecular weight).

FIG. 2A) monovalent antibodies of VL-CL-CH2-CH3 and VH-CH1-CH2-CH3 chains (as described in US 2004/0033561) and B) schematic representation of the binding of VL-CL-CH2-CH3 chains to inactive dimeric byproducts that are difficult to isolate (as described in WO 2007/048037).

FIG. 3 Biochemical characterization of MoAb c-Met (c-Met5D5MoAb ("wt")). (A) The purified antigen binding protein of protein A was isolated on a Superdex 20026/60 column. The individual peaks correspond to MoAb (3), MoAb dimer (2) and aggregate fraction (1). (B) The peak fractions (1, 2,3) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. The polyacrylamide gels were stained with coomassie blue dye.

Fig. 4 biochemical characterization of monovalent MoAb IGF1R (IGF1R AK18MoAb ("wt")). (A) The purified antigen binding protein of protein A was isolated on a Superdex 20026/60 column. The single peak corresponds to MoAb (2) and MoAb dimer (1). (B) The peak fractions (1, 2) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. The polyacrylamide gels were stained with coomassie blue dye. C) The molecular weights of peak fractions 1 and 2 were studied by SEC-MALLS. Peak 2 was identified as monovalent antigen binding protein MoAb IGF 1R.

Fig. 5 biochemical characterization of MoAb Her3(Her3205MoAb ("wt")). (A) The protein A purified antibody was isolated on a Superdex 20026/60 column. The individual peaks correspond to MoAb (3), MoAb dimer (2) and aggregate fraction (1). (B) The peak fractions (1, 2,3) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. The polyacrylamide gels were stained with coomassie blue dye.

FIG. 6 biochemical characterization of MoAbher 3(Her3205 MoAbKiH) with KiH mutation. (A) The purified antigen binding protein of protein A was isolated on a Superdex 20026/60 column. (B) The peak fractions were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. The polyacrylamide gels were stained with coomassie blue dye.

FIG. 7 biochemical characterization of MoAb IGF1R with KiH mutation (IGF1R AK18MoAb KiH). (A) The protein A purified antibody was isolated on a Superdex 20026/60 column. The single peak corresponds to MoAb (2) and MoAb dimer (1). (B) The peak fractions (1, 2) were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. The polyacrylamide gels were stained with coomassie blue dye.

FIG. 8 biochemical characterization of MoAb c-Met with KiH mutation (c-Met5D5MoAb KiH). (A) The protein A purified antibody was isolated on a Superdex 20026/60 column. (B) The peak fractions were pooled and subjected to SDS-PAGE under non-reducing and reducing conditions. The polyacrylamide gels were stained with coomassie blue dye.

FIG. 9 c-Met receptor phosphorylation assay in A549 cells. HGF was used to stimulate a549 cells in the absence or presence of c-Met binding antibody or c-Met5D5MoAb ("wt")). Total cell lysates were subjected to immunoblot analysis. The phospho-c-Met band between the 2 nonspecific bands is marked with an asterisk.

FIG. 10 cellular binding of MoAb c-Met (c-Met5D5MoAb ("wt"))) to A549 cells analyzed using flow cytometry. A549 cells were incubated with dilution series of the indicated antibodies. Bound antibodies were visualized using a secondary antibody conjugated to Fc with a fluorophore.

FIG. 11 is a schematic representation of a surface plasmon resonance assay for analyzing the binding affinity of the monovalent antigen binding protein IGF1R AK18MoAb ("wt").

FIG. 12 cellular binding of MoAb IGF-1R (IGF1R AK18MoAb ("wt")) to A549 cells analyzed using flow cytometry. A549 cells were incubated with dilution series of the indicated antibodies. Bound antibodies were visualized using a secondary antibody conjugated to Fc with a fluorophore.

Figure 13ADCC assay using a parent non-glycoengineered (non-ge) IGF1R Mab and a parent glycoengineered (ge) IGF1R Mab and a non-glycoengineered monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")). Donor-derived Peripheral Blood Mononuclear Cells (PBMCs) were incubated with prostate cancer cells (DU145) in the presence of parental non-geIGF 1R Mab (=1), parental ge IGF1R Mab (=2), and non-ge monovalent antigen binding protein IGF1R MoAb (= 3).

Figure 14 estimates internalization of IGF-1R after incubation with the parent IGF-1R IgG1 antibody and monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")), data showing that internalization of IGF-1R is reduced in potency and absolute internalization when monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) is bound.

FIG. 15 evaluation of IGF-1 induced autophosphorylation of IGF-1R after incubation with IGF-1R IgG1 antibody and monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")), the data show that IGF-1 induced autophosphorylation of IGF-1R decreases in potency when monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) is bound.

Figure 16 estimates the aggregation propensity of monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) by DLS time course experiments. No measurable increase in hydrodynamic radius (Rh) of the separated monomer fractions (see fig. 4) was detected during the 5 day period.

FIG. 17 ESI-MS spectra of monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) after deglycosylation and under non-reducing conditions.

FIG. 18 ESI-MS spectra of IGF-1R monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) after deglycosylation and reduction.

Detailed Description

The invention encompasses monovalent antigen binding proteins comprising

In a preferred embodiment of the invention, the hole can be accessed by a "tie-in hole" (")"knobs-into-holes ") (KiH) technology alters the CH3 domain of the monovalent antigen binding proteins according to the invention, as described in, for example, WO 96/027011, Ridgway, J.B., et al, Protein Eng.9(1996) 617-621; and Merchant, A.M., et al, Nat Biotechnol16(1998) 677-. In this approach, the interaction surface of the 2 CH3 domains was altered to increase heterodimerization of the 2 heavy chains containing these two CH3 domains. Each of the 2 CH3 domains (of 2 heavy chains) may be a "knot" and the other a "hole". The effect of this modification is a significant reduction in high molecular weight tetramer by-products.

Introduction of disulfide bridges further stabilizes the heterodimer (Merchant, A.M., et al, Nature Biotech16(1998) 677-.

Thus, in one aspect of the invention, the monovalent antigen binding protein is further characterized

a) The domain of CH3 of the heavy chain of the full-length antibody of b) and the CH3 domain of the modified heavy chain of the full-length antibody of b) are each in contact at an interface comprising the original interface between the antibody CH3 domains;

i) The CH3 domain of one heavy chain is altered,

And

ii) altering the CH3 domain of the other heavy chain,

Preferably, the amino acid residue with larger side chain volume is selected from arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

Preferably, the amino acid residue with a smaller side chain volume is selected from alanine (a), serine (S), threonine (T), valine (V).

In one aspect of the invention, the two CH3 domains were further altered by introducing cysteine (C) as an amino acid at the corresponding position of each CH3 domain, enabling the formation of a disulfide bridge between the two CH3 domains.

In a preferred embodiment, the monovalent antigen binding protein comprises a T366W mutation in the CH3 domain of the "junction chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "pore chain". Additional interchain disulfide bridges between the CH3 domains may also be used (Merchant, a.m., et al, Nature Biotech16(1998) 677-. Thus in another preferred embodiment, said monovalent antigen binding protein comprises a Y349C, a T366W mutation in one of the 2 CH3 domains and an E356C, a T366S, an L368A, a Y407V mutation in the other of the 2 CH3 domains, or said monovalent antigen binding protein comprises a Y349C, a T366W mutation in one of the 2 CH3 domains and a S354C, a T366S, a L368A, a Y407V mutation in the other of the 2 CH3 domains (the additional Y349C mutation in one CH3 domain and the additional E356C or S354C mutation in the other CH3 domain form an inter-chain disulfide bridge) (numbering always according to the EU index of Kabat). Alternatively or additionally, however, other tie-in hole techniques described in EP1870459a1 may also be used. Preferred examples of such monovalent antigen binding proteins are R409D in the CH3 domain of "knot chain"; the K370E mutation and D399K in the CH3 domain of "pore chain"; the E357K mutation (numbered consistently according to EU index of Kabat).

In another preferred embodiment, the monovalent antigen binding protein comprises a T366W mutation in the CH3 domain of the "knot chain" and a T366S, L368A, Y407V mutation in the CH3 domain of the "pore chain", and additionally comprises R409D in the CH3 domain of the "knot chain"; the K370E mutation and D399K in the CH3 domain of "pore chain"; the E357K mutation.

In another preferred embodiment, said monovalent antigen binding protein comprises a Y349C, T366W mutation in one of the 2 CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the 2 CH3 domains, or said monovalent antigen binding protein comprises a Y349C, T366W mutation in one of the 2 CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the 2 CH3 domains and additionally a R409D mutation in the CH3 domain of "desmodn"; the K370E mutation, and D399K in the CH3 domain of "pore chain"; the E357K mutation.

a) A modified heavy chain comprising SEQ ID NO: 1; and

b) a modified heavy chain comprising SEQ ID NO: 2.

a) A modified heavy chain comprising SEQ ID NO: 3; and

b) a modified heavy chain comprising SEQ ID NO: 4.

a) A modified heavy chain comprising SEQ ID NO: 5; and

b) a modified heavy chain comprising SEQ ID NO: 6.

a) A modified heavy chain comprising SEQ ID NO: 7; and

b) a modified heavy chain comprising SEQ ID NO: 8.

a) A modified heavy chain comprising SEQ ID NO: 9; and

b) a modified heavy chain comprising SEQ ID NO: 10.

a) A modified heavy chain comprising SEQ ID NO: 11; and

b) a modified heavy chain comprising SEQ ID NO: 12.

The term "antibody" as used herein refers to a full-length antibody consisting of two antibody heavy chains and two antibody light chains (see fig. 1). The heavy chain of a full-length antibody is a polypeptide comprising, in the N-terminal to C-terminal direction, an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1(CHI), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2(CH2), an antibody heavy chain constant domain 3(CH3), abbreviated VH-CH1-HR-CH2-CH 3; and optionally antibody heavy chain constant domain 4(CH4), in the case of an antibody of the IgE subtype. Preferably, the heavy chain of the full length antibody is a polypeptide comprising in N-terminal to C-terminal direction VH, CHI, HR, CH2 and CH 3. The light chain of a full-length antibody is a polypeptide comprising, in the N-terminal to C-terminal direction, an antibody light chain variable domain (VL), an antibody light chain constant domain (CL), abbreviated VL-CL. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Antibody chains are linked together by interpeptide disulfide bonds between the CL domain and the CH1 domain (i.e., between the light and heavy chains) and between the full-length antibody heavy chain hinge region. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g. IgG1 and IgG2), IgM, IgA, IgD and IgE. The antibody according to the invention may be from a single species, for example a human, or it may be a chimeric or humanized antibody. A full-length antibody according to the invention comprises 2 antigen-binding sites, each formed by a VH and VL pair, which both specifically bind to the same (first) antigen. The monovalent antigen binding proteins of the invention were obtained from these full-length antibodies by the following modifications: a) modifying a first heavy chain of said antibody by replacing a VH domain with a VL domain of said antibody; and b) modifying the second heavy chain of the antibody by replacing the CHI domain with the CL domain of the antibody. The resulting monovalent antigen binding protein thus contains 2 modified heavy chains and no light chain.

The C-terminus of the heavy or light chain of the full-length antibody represents the last amino acid of the C-terminus of the heavy or light chain.

The term "binding site" or "antigen binding site" as used herein denotes the region of an antigen binding protein according to the invention that actually binds to a ligand (e.g. an antigen or an antigenic fragment thereof) and which is derived from an antibody molecule or a fragment thereof (e.g. a Fab fragment). The antigen binding site according to the invention comprises an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL).

The antigen-binding site that specifically binds to the desired antigen (i.e. the VH/VL pair) may be derived from a) a known antibody to the antigen or b) a novel antibody or antibody fragment obtained by de novo synthetic immunization methods, in particular using antigenic proteins or nucleic acids or fragments thereof, or by phage display.

The antigen binding site of the monovalent antigen binding proteins of the present invention comprises 6 Complementarity Determining Regions (CDRs) that are involved in the affinity of the antigen binding site to varying degrees. There are 3 heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and 3 light chain variable domain CDRs (CDRL1, CDRL2 and CDRL 3). The extent of the CDRs and Framework Regions (FRs) was determined by comparison with a compiled database of amino acid sequences in which these regions have been defined by inter-sequence variability.

Antibody specificity refers to the selective recognition of a particular epitope by an antibody. For example, natural antibodies are monospecific. Bispecific antibodies are antibodies with 2 different antigen binding specificities. The monovalent antigen binding proteins according to the invention are "monospecific" and specifically bind to an epitope of the respective antigen.

The term "valency" as used in this application denotes the presence of a specific number of binding sites in an antibody molecule. For example, a natural antibody has 2 binding sites and is bivalent. The term "monovalent antigen binding protein" refers to a polypeptide that contains only one antigen binding site.

The full length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes. The immunoglobulin classes include the IgG, IgM, IgA, IgD and IgE classes (or isotypes) and in the case of IgG and IgA sub-classes (or subtypes) thereof. In a preferred embodiment, the full length antibody of the invention, and thus the monovalent antigen binding protein of the invention, has the constant domain structure of an antibody of the IgG class.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules consisting of a single amino acid.

The term "chimeric antibody" refers to an antibody comprising a variable, i.e., binding, region from one source or species and at least a portion of a constant region from a different source or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" encompassed by the invention are those in which the constant regions have been modified or altered relative to the constant regions of the original antibody to produce properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such chimeric antibodies are also known as "class-switched antibodies". Chimeric antibodies are the expression product of an immunoglobulin gene comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for producing chimeric antibodies include conventional recombinant DNA and gene transfection techniques, which are well known in the art. See, e.g., Morrison, S.L., et al, Proc.NatI.Acad.Sci.USA81(1984) 6851-6855; US5,202,238 and US5,204,244.

The term "humanized antibody" refers to an antibody in which the framework or "complementarity determining region" (CDR) has been modified compared to the framework or complementarity determining region of the parent immunoglobulin to comprise CDRs of the immunoglobulin of different specificity. In a preferred embodiment, murine CDRs are grafted into the framework regions of a human antibody to make a "humanized antibody". See, e.g., Riechmann, L., et al, Nature332(1988) 323-327; and Neuberger, M.S., et al, Nature314(1985)268- & 270. Particularly preferred CDRs correspond to those sequences described for the above antigens that recognize the chimeric antibody. Other forms of "humanized antibodies" encompassed by the invention are those in which the constant regions have been additionally modified or altered relative to the original constant region antibody to produce properties according to the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. Human antibodies are well known in the art (van Dijk, m.a., and van de Winkel, j.g., curr. opin. chem. biol.5(2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that, upon immunization, are capable of producing a complete repertoire of human antibodies or selected human antibodies in the absence of endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays in these germline mutant mice will result in the production of human antibodies following antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA90(1993) 2551-2555; Jakobovits, A., et al, Nature362(1993) 255-258; Bruggemann, M., et al, Yeast Immunol.7(1993) 33-40). Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G., J.Mol.biol.227(1992) 381-. Human Monoclonal antibodies can also be prepared using the techniques of Cole et al and Boerner et al (Cole, et al, Monoclonal antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); and Boerner, P.et al, J.Immunol.147(1991) 86-95). As already mentioned in the context of the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also encompasses antibodies which have been modified in the constant region to produce the properties according to the invention, in particular with regard to C1q binding and/or FcR binding, for example by "class switching", i.e. by altering or mutating the Fc part (e.g. from IgG1 to IgG4 and/or IgG 1/IgG 4 mutations).

The term "recombinant human antibody" as used herein is intended to include all human antibodies prepared, expressed, produced or isolated by recombinant means, e.g., antibodies isolated from a host cell, e.g., NS0 or CHO cells or from a transgenic animal (e.g., mouse) for human immunoglobulin genes, or antibodies expressed using a recombinant expression vector transfected into a host cell. Such recombinant human antibodies have variable and constant regions in rearranged form. Recombinant human antibodies according to the invention have been hypermutated in vivo in somatic cells. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

As used herein, "variable domain" (light chain variable domain (VL), heavy chain variable domain (VH)) means each light and heavy chain pair directly involved in binding of an antibody to an antigen. The domains of variable human light and heavy chains have the same general structure, and each domain comprises 4 widely conserved Framework (FR) regions of sequence connected by 3 "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation and the CDRs may form loops connecting the β -sheet structures. The CDRs in each chain maintain their three-dimensional structure through the framework regions and form together with the CDRs from the other chains the antigen binding site. The antibody heavy and light chain CDR3 regions play a particularly important role in the binding specificity/affinity of the antibodies according to the invention and therefore provide another object of the invention.

As used herein, the term "hypervariable region" or "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions comprise amino acid residues from "complementarity determining regions" or "CDRs". "framework" or "FR" regions are those variable domain regions that are not hypervariable region residues as defined herein. Thus, the light and heavy chains of an antibody comprise, from N-to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The CDRs on each chain are separated by these framework amino acids. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding. CDR and FR regions were determined according to the standard definition of Kabat, et al, Sequences of Proteins of Immunological Interest, fifth edition, public Health Service, National Institutes of Health, Bethesda, Md. (1991).

The term "binding" or "specific binding" as used herein refers to the binding of a monovalent antigen binding protein to an antigenic epitope in an in vitro assay, preferably in a plasma resonance assay (BIAcore, GE-healthcare uppsala, sweden) using purified wild-type antigen. By the term ka (rate constant for association of antibody from antibody/antigen complex), k_D(dissociation constant) and K_D(k_D/ka) defines the binding affinity. Binding or specific binding means 10^-8mol/l or less, preferably 10^-9M to 10^-13Binding affinity (K) in mol/l_D). Thus, a monovalent antigen binding protein according to the invention specifically binds to each of the antigens with a binding specificity of 10^-8mol/l or less, preferably 10^-9M to 10^-13Binding affinity (K) in mol/l_D) The specific antigen of (1).

Binding of monovalent antigen binding proteins to Fc γ RIII can be studied by BIAcore assay (GE-Healthcare Uppsala, sweden). By the term ka (rate constant for association of antibody from antibody/antigen complex), k_D(dissociation constant) and K_D(k_D/ka) defines the binding affinity.

The term "epitope" includes any polypeptide determinant capable of specific binding to a monovalent antigen binding protein. In certain embodiments, epitope determinants include chemically active surface groups of the molecule, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, and in certain embodiments, epitope determinants may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by a monovalent antigen binding protein.

In certain embodiments, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

In other embodiments, the monovalent antigen binding protein according to the invention is characterized in that the full length antibody is of the subclass human IgG1, or human IgG1 with mutations L234A and L235A.

In other embodiments, the monovalent antigen binding protein according to the invention is characterized in that said full length antibody is of the human IgG2 subclass.

In other embodiments, the monovalent antigen binding protein according to the invention is characterized in that said full length antibody is of the human IgG3 subclass.

In other embodiments, the monovalent antigen binding protein according to the invention is characterized in that the full length antibody is of the human IgG4 subclass, or of the human IgG4 subclass with the additional mutations S228P and L235E (also known as IgG4 SPLE).

The term "constant region" is used in this application to denote the sum of domains of an antibody of the non-variable region. The constant region is not directly involved in antigen binding, but exhibits multiple effector functions. Depending on the amino acid sequence of its heavy chain constant region, antibodies are classified (also called isotypes): IgA, IgD, IgE, IgG and IgM, some of these classes can be further divided into subclasses (also called isotypes), such as IgG1, IgG2, IgG3 and IgG4, IgA1 and IgA 2. The heavy chain constant regions corresponding to the different antibody types are referred to as α, δ: ε, γ and μ. The light chain constant regions (CL) that can be found in all 5 antibody types are called kappa (kappa) and lambda (lambda).

The term "constant region derived from a human source" is used in the present application to denote the constant heavy chain region and/or constant light chain kappa or lambda region of a human antibody of the subclass IgG1, IgG2, IgG3, or IgG 4. Such constant regions are well known in the art, as described, for example, in Kabat, E.A. (see, e.g., Johnson, G. and Wu, T.T., Nucleic Acids Res.28(2000) 214-.

While antibodies of the IgG4 subclass showed reduced Fc receptor (Fc γ RIIIa) binding, antibodies of other IgG subclasses showed strong binding. However, reduced Fc receptor binding is also provided if residues Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434 and His435 are altered (Shields, R.L., et al, J.biol.chem.276(2001) 6591-6604; Lund, J., et al, FASEB J.9(1995) 115-119; Morgan, A., et al, Immunology86(1995) 319-324; EP 0307434).

In one embodiment, the antibody according to the invention has reduced FcR binding compared to the IgG1 antibody, and the full length parent antibody is associated with FcR binding of the IgG4 subclass or of the IgG1 or IgG2 subclass, has S228, L234, L235 and/or D265 mutations, and/or comprises a PVA236 mutation. In one embodiment, the mutation in the full-length parent antibody is S228P, L234A, L235A, L235E and/or PVA 236. In another embodiment, the mutations in the full-length parent antibody are S228P and L235E in IgG4 and L234A and L235A in IgG 1.

The constant regions of antibodies are directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by the binding of complement factor C1q to the constant regions of most IgG antibody subclasses. The binding of C1q to antibodies is caused by defined protein-protein interactions at the so-called binding sites. Such constant region binding sites are known in the art and described, for example, in Lukas, T.J., et al, J.Immunol.127(1981) 2555-2560; bunkhouse, r. and Cobra, j.j., mol.immunol.16(1979) 907-; burton, D.R., et al, Nature288(1980) 338-344; thomason, J.E., et al, mol.Immunol.37(2000) 995-; idiocies, e.e., et al, j.immunol.164(2000) 4178-; hearer, M., et al, J.Virol.75(2001) 12161-; morgan, A., et al, Immunology86(1995)319- "324; and EP 0307434. These constant region binding sites are for example characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat).

The term "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to the lysis of human target cells by an antibody according to the invention in the presence of effector cells. ADCC is preferably measured by treating the antigen expressing cell preparation with an antibody according to the invention in the presence of effector cells, e.g. freshly isolated PBMCs or purified effector cells from buffy coat, such as monocytes or Natural Killer (NK) cells or permanently growing NK cell lines.

It has surprisingly been found that the antigen binding protein according to the invention shows improved ADCC performance compared to its parent full length antibody. These improved ADCC effects are achieved without further modification of the Fc part, e.g. glycoengineering. The term "Complement Dependent Cytotoxicity (CDC)" refers to the process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. The binding of C1q to antibodies is caused by defined protein-protein interactions at the so-called binding sites. Such Fc moiety binding sites are known in the art (see above). These Fc moiety binding sites are characterized, for example, by amino acids L234, L235, D270, N297, E318, K320, K322, P331 and P329 (numbering according to EU index of Kabat). Antibodies of the IgG1, IgG2, and IgG3 subclasses typically show complement activation including C1q and C3 binding, while IgG4 does not activate the complement system and also does not bind C1q and/or C3.

The cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide components as described in Umana, P.et al, Nature Biotechnol.17(1999)176-180 and U.S. Pat. No. 6,602,684. The most commonly used therapeutic antibody, the IgG 1-type antibody, is a glycoprotein with a conserved N-linked glycosylation site at Asn297 in each CH2 domain. The 2 complex, bisected oligosaccharides attached to Asn297 are buried between the CH2 domains, form a large number of contacts with the polypeptide backbone, and their presence is critical for antibody-mediated effector functions, such as antibody-dependent cell-mediated cytotoxicity (ADCC) (Life, M.R., et al, Glycobiology5(1995) 813-822; Jefferis, R., et al, Immunol.Rev.163(1998) 59-76; Wright, A., and Morrison, S., L., Trends Biotehneol.15 (1997) 26-32). Umana, P.et al Nature Biotechnol.17(1999)176-180 and WO99/54342 show that overexpression of β (1, 4) -N-acetylglucosaminyltransferase III ("GnTIII"), a glycosyltransferase that catalyzes the formation of a di-branched oligosaccharide, in Chinese Hamster Ovary (CHO) cells significantly increases the in vitro ADCC activity of the antibody. Modification of Asn297 carbohydrate composition or elimination thereof also affects binding to Fc γ R and C1q (Umana, P., et al, Nature Biotechno1.17(1999) 176-.

In one aspect of the invention, the monovalent antigen binding protein according to the invention is characterized in that the modified heavy chains of a) and b) are of the IgG1 type and the antigen binding protein is afucosylated, having an amount of fucose of 80% or less of the total amount of oligosaccharides (sugars) at Asn 297.

In one embodiment, the antigen binding protein is an afucosylated with an amount of fucose of 65% to 5% of the total amount of oligosaccharides (sugars) at Asn 297.

The term "afucosylated antigen binding protein" refers to an antigen binding protein of the IgG1 or IgG3 isotype (preferably the IgG1 isotype) with a reduced level of fucose residues at position Asn297 of the Fc region with an altered glycosylation pattern. Glycosylation of human IgG1 or IgG3 occurred at position Asn297, with the two-branched complex oligosaccharide being core fucosylated being glycosylated and terminating with up to 2 Gal residues. Depending on the amount of terminal Gal residues, these structures were designated as G0, G1(α 1,6 or α 1, 3) or G2 glycan residues (Raju, t.s., BioProcess int.1(2003) 44-53). CHO-type glycosylation of the Fc part of antibodies is described, for example, in Router, F.H., Glycoconjugate J.14(1997) 201-207. Antibodies recombinantly expressed in a CHO host cell without sugar modification are typically fucosylated at position Asn297 in an amount of at least 85%. It is to be understood that the term afucosylated antibody as used herein includes antibodies without fucose in their glycosylation pattern. It is well known that the typical residue position for glycosylation in antibodies is asparagine ("Asn 297") at position 297 according to the EU numbering system.

Thus, an afucosylated antigen binding protein according to the invention denotes an antibody of IgG1 or IgG3 isotype, preferably IgG1 isotype, wherein the amount of fucose is 80% or less (e.g. 80% to 1%) of the total amount of oligosaccharides (sugars) at position Asn297 (which means that at least 20% or more of the oligosaccharides at position Asn297 in the Fc region are afucosylated). In one embodiment, the amount of fucose is 65% or less (e.g., 65% to 1%), in one embodiment, from 65% to 5%, and in one embodiment, from 40% to 20% of the oligosaccharides at position Asn297 of the Fc region. According to the present invention, "amount of fucose" means the amount of said oligosaccharide (fucose) within the oligosaccharide (sugar) chain at position Asn297, relative to the sum of all oligosaccharides (sugars) attached to Asn297 (e.g. complex, mixed and high mannose structures), said amount being measured by MALDI-TOF mass spectrometry and calculated as a mean value (see e.g. WO2008/077546 for detailed procedures for determining the amount of fucose). Furthermore, in one embodiment, the oligosaccharides of the Fc region are di-branched. The afucosylated antibodies according to the invention may be expressed in a carbohydrate-modified host cell engineered to express at least one nucleic acid encoding a polypeptide with GnTIII activity in an amount sufficient to partially fucosylate oligosaccharides in the Fc region. In one embodiment, the polypeptide having GnTIII activity is a fusion polypeptide. Alternatively, the α 1, 6-fucosyltransferase activity of the host cell may be reduced or eliminated according to US6,946,292 to produce a sugar-modified host cell. The amount of antibody fucosylation can be predetermined, for example, by fermentation conditions (e.g., fermentation time) or by combining at least 2 antibodies with different amounts of fucosylation. Such afucosylated antigen-binding proteins and corresponding methods of glycoengineering are described in WO2005/044859, WO2004/065540, WO2007/031875, Umana, P., et al, Nature Biotechnol.17(1999)176-180, WO99/154342, WO2005/018572, WO2006/116260, WO2006/114700, WO2005/011735, WO2005/027966, WO97/028267, US2006/0134709, US2005/0054048, US2005/0152894, WO2003/035835, WO 2000/061739. Glycoengineered antigen binding proteins according to the invention have increased ADCC (compared to the parent antigen binding protein). Other methods of glycoengineering to produce afucosylated antigen binding proteins according to the invention are described in, for example, Niwa, r.et al, j.immunol.methods306(2005) 151-160; shinkawa, t., et al, j.biol.chem, 278(2003) 3466-; WO03/055993 or US 2005/0249722.

One aspect of the invention is therefore an afucosylated antigen binding protein according to the invention for use in the treatment of cancer, which is of the IgG1 isotype or of the IgG3 isotype, preferably of the IgG1 isotype, with an amount of fucose of 60% or less (e.g. 60% to 1%) of the total amount of oligosaccharides (sugars) at the Ash 297. Another aspect of the invention is the use of an afucosylated anti-CD 20 antibody specifically binding to the IgG1 or IgG3 isotype (preferably IgG1 isotype) of CD20, having an amount of fucose of 60% or less of the total amount of oligosaccharides (sugars) at Ash297, for the manufacture of a medicament for the treatment of cancer. In one embodiment, the amount of fucose is between 60% and 20% of the total amount of oligosaccharides (sugars) at position Asn 297. In one embodiment, the amount of fucose is between 60% and 40% of the total amount of oligosaccharides (sugars) at position Asn 297. In one embodiment, the amount of fucose is between 0% of the total amount of oligosaccharides (sugars) at position Asn 297.

When referring to residues or positions in the constant region of an immunoglobulin heavy chain, the "EU numbering system" or "EU index (according to Kabat)" is generally used (e.g., EU index is reported in Kabat et al, sequential Proteins of Immunological Interest, fifth edition, Public Health Service, National Institutes of Health, Bethesda, MD (1991), which is expressly incorporated herein by reference).

The term "sugar chain showing the characteristics of an N-linked glycan attached to Asn297 of an antibody recombinantly expressed in CHO cells" means that the sugar chain at position Asn297 of the full length parent antibody according to the invention has, apart from the fucose residue, the same structure and sugar residue sequence as the same antibody expressed in unmodified CHO cells (e.g. the antibody reported in WO 2006/103100).

The term "NGNA" as used in this application denotes the sugar residue N-glycolylneuraminic acid.

The antibodies according to the invention are produced by recombinant means. Thus, one aspect of the invention is a nucleic acid encoding an antibody according to the invention, and another aspect is a cell comprising said nucleic acid encoding an antibody according to the invention. Methods of recombinant production are well known in the art and include protein expression in prokaryotic and eukaryotic cells, and subsequent isolation and usually purification of the antibody to a pharmaceutically acceptable purity. To express the above antibodies in host cells, nucleic acids encoding the respective modified light and heavy chains are inserted into expression vectors by standard methods. Expression is carried out in suitable prokaryotic or eukaryotic host cells such as CHO cells, NS0 cells, SP2/0 cells, HEK293 cells, COS cells, PER. C6 cells, yeast or E.coli cells, and the antibody is recovered from the cells (supernatant or lysed cells). General methods for recombinant production of antibodies are well known in the art and are described, for example, in Makrides, S.C., Protein Expr. Purif.17(1999) 183-202; geisse, S., et al, Protein Expr. Purif.8(1996) 271-282; kaufman, R.J., mol.Biotechnol.16(2000) 151-161; werner, R.G., Drug Res.48(1998) 870-.

The monovalent antigen binding proteins according to the invention are suitably isolated from the culture medium by conventional immunoglobulin purification procedures, such as protein a-sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis or affinity chromatography.

DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells serve as a source of such DNA and RNA. Once isolated, the DNA can be inserted into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulins, such as HEK293 cells, CHO cells, or myeloma cells, to obtain synthesis of recombinant monoclonal antibodies in the host cells.

Amino acid sequence variants (or mutants) of monovalent antigen binding proteins are prepared by introducing appropriate nucleotide changes in the antibody DNA, or by nucleotide synthesis. However, such modifications can only be made within a very limited range such as described above. For example, the modifications do not alter the above-described antibody characteristics (e.g., IgG isotype and antigen binding), but may improve yield of recombinant production, protein stability, or aid in purification.

The term "host cell" as used in the present application denotes any type of cell system that can be engineered to produce an antibody according to the invention. In one embodiment, HEK293 cells and CHO cells are used as host cells. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary such cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as used to screen the original transformed cell are also included.

For example, Barnes, L.M., et al, Cytotechnology32(2000)109- "123; barnes, L.M., et al, Biotech.Bioeng.73(2001)261-270 describe expression in NS0 cells. Transient expression is described, for example, by Durocher, Y., et al, Nucl. acids. Res.30(2002) E9. Orlandi, R.et al, Proc.Natl.Acad.Sci.USA86(1989) 3833-3837; carter, p., et al, proc.natl.acad.sci.usa89(1992) 4285-; and Norderhaug, l., et al, j.immunol 1.methods204(1997)77-87, describe the cloning of variable domains. Schlaeger, E.J., and Christensen, K., Cytology 30(1999) 71-83 and Schlaeger, E.J., J.Immunol. Methods194(1996) 191-199 describe preferred transient expression systems (HEK 293).

Suitable control sequences for prokaryotic cells include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, enhancers and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a presequence or secretory leader DNA is operably linked to polypeptide DNA if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or is operably linked to a coding sequence if the position of the ribosome binding site facilitates translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation is accomplished by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Purification of monovalent antigen binding proteins to remove cellular components or other contaminants, such as other cellular nucleic acids or proteins (e.g., by-products), is performed by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art (see Ausubel, f., et al (eds.), Current Protocols in Molecular Biology, greene publishing and Wiley inter science, New York (1987)). Different methods for protein purification are well established and widely used, such as affinity chromatography using microbial proteins (e.g. protein a or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed mode exchange), thiophilic adsorption (e.g. using β -mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. using phenyl sepharose, aza-arenophilic resin or metaaminophenylboronic acid), metal chelator affinity chromatography (e.g. using ni (ii) and cu (ii) affinity materials), size exclusion chromatography and electrophoresis methods (e.g. gel electrophoresis, capillary electrophoresis) (vijayaakshi, m.a., appl.biochem.biotech.75(1998) 93-102). Examples of purification are described in example 1 and corresponding figures 3 to 8.

One aspect of the invention is a pharmaceutical composition comprising an antibody according to the invention. Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a pharmaceutical composition. Another aspect of the invention is a method of manufacturing a pharmaceutical composition comprising an antibody according to the invention. In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an antibody according to the invention formulated with a pharmaceutical carrier.

One embodiment of the invention is a monovalent antigen binding protein according to the invention for use in the treatment of cancer.

Another aspect of the invention is said pharmaceutical composition for use in the treatment of cancer.

Another aspect of the invention is the use of an antibody according to the invention for the manufacture of a medicament for the treatment of cancer.

Another aspect of the invention is a method of treating a patient suffering from cancer by administering an antibody according to the invention to a patient in need of such treatment.

As used herein, "pharmaceutical carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and delayed absorption agents, and the like that are physiologically compatible. Preferably, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).

The compositions of the present invention can be administered by a variety of methods known in the art. The skilled artisan will appreciate that the route and/or mode of administration will vary depending on the desired result. In order to administer a compound of the invention by some route of administration, it may be necessary to coat the compound with a material that avoids its inactivation, or to co-administer said material with a chemotherapeutical agent, e.g., a compound that can be administered to a subject in a suitable carrier, e.g., a liposome or diluent. Pharmaceutically acceptable diluents include saline and aqueous buffer solutions. Pharmaceutical carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. The use of such media and active agents for pharmaceutically active substances is known in the art.

The phrases "parenteral administration" and "parenterally administered" as used herein refer to modes of administration other than enteral and topical administration, typically by injection, including, but not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

The term cancer as used herein refers to a proliferative Disease, such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchioloalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer (stomach cancer), stomach cancer (gastic cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the esophagus, cancer of the small intestine, cancer of the endocrine system, cancer of the thyroid gland, cancer of the parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, cancer of the prostate, cancer of the bladder, cancer of the kidney or ureter, cancer of the renal cell, cancer of the kidney, mesothelioma, hepatocellular carcinoma, cancer of the biliary tract, Central Nervous System (CNS) tumor, Spinal axis tumors, brain stem gliomas, glioblastoma multiforme, astrocytomas, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and ewing's sarcoma (Ewingssarcoma), including refractory forms of any of the foregoing cancers, or combinations of one or more of the foregoing cancers.

These compositions may also contain adjuvants such as preserving, wetting, emulsifying, and dispersing agents. Prevention of the presence of microorganisms can be ensured by the above sterilization procedures and the addition of various antibacterial and antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to include isotonic agents, for example, sugars, sodium chloride, and the like in the compositions. In addition, prolonged absorption of the injectable pharmaceutical form can be brought about by the addition of active agents which delay absorption, for example, aluminum monostearate and gelatin.

Regardless of the route of administration chosen, the compounds of the invention, which may be used in a suitable hydrated form, and/or the pharmaceutical compositions of the invention, are formulated into pharmaceutically acceptable dosage forms by conventional methods known to those skilled in the art.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular composition of the invention employed, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, body weight, condition, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

The composition must be sufficiently sterile and fluid so that the composition can be delivered by syringe. In addition to water, the carrier is preferably an isotonic buffered saline solution.

Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol in the composition and sodium chloride.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably and all such designations include progeny. Thus, the words "transformant" and "transformed cell" include primary such cells and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be exactly identical in DNA content, due to deliberate or inadvertent mutations. Variant progeny that have the same function or biological activity as used to screen the original transformed cell are also included. When different names are indicated, it will be apparent from the context.

The term "transformation" as used herein refers to the process of transferring a vector/nucleic acid into a host cell. If cells without difficult-to-surmount cell wall barriers are used as host cells, transfection can be carried out by the calcium phosphate precipitation method described, for example, by Graham and Van der Eh, Virology52(1978) 546. However, other methods of introducing DNA into cells may also be used, for example by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing a large number of cell wall structures are used, one method of transfection is, for example, calcium treatment with calcium chloride as described by Cohen, F.N, et al, PNAS.69(1972) 7110.

As used herein, "expression" refers to the process of transcription of a nucleic acid into mRNA and/or the subsequent translation of the transcribed mRNA (also known as transcript) into a peptide, polypeptide or protein. The transcripts and the encoded polypeptides are collectively referred to as gene products. If the polynucleotide is derived from genomic DNA, expression in a eukaryotic cell may include splicing of the mRNA.

A "vector" is a nucleic acid molecule, particularly a self-replicating nucleic acid molecule, which transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors whose primary function is to insert DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors whose primary function is to replicate DNA or RNA, and expression vectors whose function is to transcribe and/or translate DNA or RNA. Also included are vectors that provide more than one of the above functions.

An "expression vector" is a polynucleotide that, when introduced into a suitable host cell, is transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell comprised of an expression vector that can function to produce a desired expression product.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is to be understood that modifications may be made to the procedures set forth without departing from the spirit of the invention.

Sequence listing description

SEQ ID NO: 1c-Met 5D5MoAb ("wt") -modified heavy chain a) VL-CH1-CH2-CH3

SEQ ID NO: 2c-Met 5D5MoAb ("wt") -modified heavy chain b) VH-CL-CH2-CH3

SEQ ID NO: 3IGF1R AK18MoAb ("wt") -modified heavy chain a) VL-CH1-CH2-CH3

SEQ ID NO: 4IGF1R AK18MoAb ("wt") -modified heavy chain b) VH-CL-CH2-CH3

SEQ ID NO: 5Her 3205MoAb ("wt") -modified heavy chain a) VL-CH1-CH2-CH3

SEQ ID NO: 6Her 3205MoAb ("wt") -modified heavy chain b) VH-CL-CH2-CH3

SEQ ID NO: 7c-Met 5D5MoAb KiH modified heavy chain a) VL-CH1-CH2-CH3 junction T366W, S354C

SEQ ID NO: 8c-Met 5D5MoAb KiH modified heavy chain b) VH-CL-CH2-CH3 pore L368A, Y407V, T366S, Y349C

SEQ ID NO: 9IGF1R AK18MoAb KiH modified heavy chain a) VL-CH1-CH2-CH3 node T366W, S354C

SEQ ID NO: 10IGF1R AK18MoAb KiH modified heavy chain b) VH-CL-CH2-CH3 pore L368A, Y407V, T366S, Y349C

SEQ ID NO: 11Her 3205MoAb KiH modified heavy chain a) VL-CH1-CH2-CH3 junction T366W, S354C

SEQ ID NO: 12Her 3205MoAb KiH modified heavy chain b) VH-CL-CH2-CH3 well L368A, Y407V, T366S, Y349C

Experimental procedures

A. Materials and methods:

recombinant DNA technology

Such as Sambrook, j, et al, Molecular cloning: a laboratory manual; DNA manipulation was performed using standard methods as described in Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1989). Molecular biological reagents were used according to the manufacturer's instructions.

DNA and protein sequence analysis and sequence data management

General information on the nucleotide Sequences of the light and heavy chains of human immunoglobulins is provided in Kabat, E.A. et al, (1991) Sequences of Proteins of Immunological Interest, fifth edition, NIHPublication No 91-3242. The amino acids of the antibody chains were numbered according to EU numbering (Edelman, G.M., et al, PNAS63(1969) 78-85; Kabat, E.A., et al (1991) Sequences of Proteins of immunological Interest, fifth edition, NIH Publication No 91-3242). GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and Infmax's Vector NTI Advance software Group version 8.0 were used for sequence generation, mapping, analysis, annotation and specification.

DNA sequencing

The DNA sequence was determined by double-strand sequencing performed in SequiServe (Vaterstetten, Germany) and Geneart AG (Regensburg, Germany).

Gene synthesis

The desired gene segments were prepared from synthetic oligonucleotides and PCR products by automated gene synthesis from Geneart AG (Regensburg, germany). The gene segment flanked by a single restriction enzyme cleavage site was cloned into the pGA18(ampR) plasmid. Plasmid DNA was purified from the transformed bacteria and the concentration was determined by UV spectroscopy. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. A complete fragment of the DNA sequence encoding 2 antibody chains (VH-CL-CH2-CH3 and VL-CH1-CH2-CH3) flanked by 5'HpaI and 3' NaeI restriction sites was prepared by gene synthesis. Gene segments encoding "binding-in-hole" were synthesized with 5'-BclI and 3' -NaeI restriction sites, indicating that one antibody heavy chain carries the T366W mutation in the CH3 domain and the second antibody heavy chain carries the T366S, L368A and Y407V mutations in the CH3 domain. In a similar manner, DNA sequences encoding "binding-in holes" flanked by BclI and NaeI restriction sites were prepared by gene synthesis, i.e. the antibody heavy chain carries the S354C and T366W mutations in the CH3 domain and the second antibody heavy chain carries the Y349C, T366S, L368A and Y407V mutations. All constructs were designed with a 5' -terminal DNA sequence encoding a leader peptide that targets secretion of the protein in eukaryotic cells.

Construction of expression plasmids

All antibody chains were constructed using Roche expression vector. The carrier consists of the following elements:

-origin of replication, oriP, of Epstein-Barr Virus (EBV),

the origin of replication of the pUC18 vector, which allows the replication of this plasmid in E.coli

-a beta-lactamase gene, which confers ampicillin resistance in E.coli,

immediate early enhancer and promoter from Human Cytomegalovirus (HCMV),

-a human 1-immunoglobulin polyadenylation ("poly a") signal sequence, and

unique HpaI, BclI and NaeI restriction sites.

Immunoglobulin genes and "tie-in" constructs in the order VH-CL-CH2-CH3 and VL-CH1-CH2-CH3 were prepared by gene synthesis as described above and cloned into pGA18(ampR) plasmid. pG18(ampR) plasmid carrying the synthesized DNA segment and the Roche expression vector were digested with HpaI and NaeI or with BclI and NaeI restriction enzymes (Roche molecular Biochemicals) and subjected to agarose gel electrophoresis. The purified DNA segment was then ligated into the isolated Roche expression vector HpaI/NaeI or BclI/NaeI fragment to generate the final expression vector. The final expression vector was transformed into E.coli cells, expression plasmid DNA (miniprep) was isolated and restriction enzyme analysis and DNA sequencing were performed. The correct clones were cultured in 150ml LB-Amp medium, and plasmid DNA (Maxiprep) was re-isolated and sequence integrity verified by DNA sequencing.

Transient expression of immunoglobulin variants in HEK293 cells

Using FreeStyle^TM293 expression System, recombinant immunoglobulin variants were expressed by transient transfection of human embryonic kidney 293-F cells according to the manufacturer's instructions (Invitrogen, USA). Briefly, in FreeStyle^TM293 expression Medium at 37 ℃/8% CO₂Under-culture suspended FreeStyle^TM293-F cells. 1-2X10 on the day of transfection⁶Viable cells/ml the cells were seeded in fresh medium. In thatPreparation of DNA-293fectin I Medium (Invitrogen, USA)^TMComplexes using 325. mu.l 293fectin^TM(Invitrogen, Germany) and 1: a1 molar ratio of 250. mu.g of each plasmid DNA was used for a final transfection volume of 250 ml. Cell culture supernatants containing the antibodies were harvested 7 days post-transfection by centrifugation at 14000g for 30 minutes and filtration through sterile filters (0.22 μm). The supernatant was stored at-20 ℃ until purification.

Alternatively, antibodies were generated by transient transfection in HEK293-EBNA cells. HEK293-EBNA cells grown adherent by culturing in DMEM (Dulbecco's modified Eagle medium, Gibeo) supplemented with 10% ultra-low IgG FCS (fetal calf serum, Gibco), 2mM L-glutamine (Gibco) and 250 μ g/ml geneticin (Gibco) (human embryonic kidney cell line 293 expressing Epstein-Barr virus nuclear antigen;american type culture Collection number ATCC # CRL-10852, Lot.959218) were transiently co-transfected with each expression plasmid to express the antibody. For transfection, the ratio of 4: FuGENE of 1 (ranging from 3: 1 to 6: 1)^TMFuGENE was used at a ratio of reagent (. mu.l) to DNA (. mu.g)^TM6 transfection reagent (Roche Molecular Biochemicals). Proteins were expressed from the respective plasmids using equimolar proportions of the plasmids. L-Glutamine ad4mM, glucose [ Sigma ] on day 3]And NAA [ Gibco ]]Feeder cells. Cell culture supernatants containing bispecific antibody were harvested by centrifugation from day 5 to 11 post transfection and stored at-20 ℃. General information on recombinant expression of human immunoglobulins in, for example, HEK293 cells is provided in Meissner, P.et al, Biotechnol.Bioeng.75(2001) 197-203.

Purification of antibodies

Using protein A-agarose^TM(GE Healthcare, Sweden) and Superdex200 size exclusion chromatography antibodies were purified from cell culture supernatants by affinity chromatography. Briefly, sterile filtered cell culture supernatant was applied to cells in PBS buffer (10mM Na)₂HPO₄，1mM KH₂PO₄137mM NaCl and 2.7mM KCl, pH7.4) in a 5ml HiTrap protein A HP column. Unbound protein was washed off with equilibration buffer. The antibodies and antibody variants were eluted with 0.1M citrate buffer, pH2.8, and the protein-containing fractions were neutralized with 0.1ml1M Tris, pH 8.5. The eluted protein fractions were then pooled, concentrated to a volume of 3ml using an Amicon ultracentrifugal filtration unit (MWCO: 30K, Millipore) and loaded onto Superdex200HiLoad120ml16/60 or 26/60 gel filtration columns (GEHealthcare, Sweden) equilibrated with 20mM histidine, 140mM NaCl, pH 6.0. Fractions containing purified antibody with less than 5% high molecular weight aggregates were combined and stored at-80 ℃ in 1.0mg/ml aliquots.

Analysis of purified proteins

Protein concentration of the purified protein samples was determined by measuring the Optical Density (OD) at 280am using molar extinction coefficients calculated based on amino acid sequence. By the presence of orThe purity and molecular weight of the antibodies were analyzed by SDS-PAGE in the absence of reducing agent (5mM1, 4-dithiothreitol) and stained with Coomassie Brilliant blue. Used according to the manufacturer's instructionsPre-gel systems (Invitrogen, USA) (4-12% Tris-glycine gels). At 25 ℃ at 200mMKH₂PO₄The aggregate content of the antibody samples was analyzed by high performance SEC using a Superdex200 analytical size exclusion column (GE Healthcare, sweden) in 250mM KCl, ph7.0 running buffer. 25 μ g of protein was injected into the column at a flow rate of 0.5 ml/min and eluted isocratically for 50 minutes. For stability analysis, the purified protein at a concentration of 1mg/ml was incubated at 4 ℃ and 40 ℃ for 7 days, then evaluated by high performance SEC (e.g., HP SEC analysis (purified protein).) after removal of N-glycans by enzymatic treatment with peptide-N-glycosidase f (roche Molecular biochemicals), the integrity of the amino acid backbone of the light and heavy chains of the reduced bispecific antibody was confirmed by NanoElectrospray Q-TOF mass spectrometry.

Mass Spectrometry and SEC-MALLS

Mass spectrometry

The mass of total deglycosylation of the antibody was determined and confirmed by electrospray ionization mass spectrometry (ESI-MS). Briefly, 100. mu.g of purified antibody at a protein concentration of up to 2mg/ml was deglycosylated with 50mU of N-glycosidase F (PNGaseF, ProZyme) in 100mM KH2PO4/K2HPO4, pH7 at 37 ℃ for 12-24 hours and subsequently desalted by HPLC on a Sephadex G25 column (GEHealthcare). The mass of each heavy and light chain was determined by ESI-MS after deglycosylation and reduction. Briefly, 115. mu.l of 50. mu.g antibody was incubated with 60. mu.l of 1M TCEP and 50. mu.l of 8M guanidine hydrochloride, followed by desalting. The total mass and mass of the reduced heavy and light chains were determined by ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source. The mass range recorded depends on the sample molecular weight. In general, the mass range is set from 600-2000m/z for reduced antibodies and from 1000-3600m/z for non-reduced antibodies or bispecific molecules.

SEC-MALLS

The approximate molecular weight of the proteins in solution was determined using SEC-MALLS (size exclusion chromatography with multi-angle laser light scattering). According to light scattering theory, MALLS allows assessment of the molecular weight of macromolecules, regardless of their molecular behavior or other assumptions. SEC-MALLS is based on the separation of proteins according to their size (hydrodynamic radius) by SEC chromatography and subsequent concentration and scattered light sensitive detectors. SEC-MALLS generally yields molecular weight estimates that allow for the precision of clearly resolving monomers, dimers, trimers, etc., provided that SEC separation is sufficient.

The following instruments were used in this work: dionex Ultimate3000 HPLC; column: superose610/300(GE Healthcare); eluent: 1x PBS; flow rate: 0.25 mL/min; a detector: OptiLab REX (Wyatt inc., Dernbach), MiniDawn Treos (Wyatt inc., Dernbach). Molecular weights were calculated using Astra software, version 5.3.2.13. Between 50 and 150 μ g of protein mass was loaded onto the column and bsa (sigma aldrich) was used as reference protein.

Dynamic Light Scattering (DLS) time course

Samples (30. mu.L) at a concentration of about 1mg/mL in 20mM His/HisCl, 140mM NaCl, pH6.0 were filtered through 384-well filter plates (0.45 μm well size) into 384-well optical plates (Corning) and covered with 20 μ L paraffin oil (Sigma). Dynamic light scattering data was collected repeatedly using a DynaPro DLS plate reader (Wyatt) at a constant temperature of 40 ℃ over a5 day period. Data were processed with dynamicv6.10 (Wyatt).

c-Met phosphorylation assay

The day before HGF stimulation, 5x10e 5a 549 cells were seeded in RPMI with 0.5% FCS (fetal calf serum) in each well of a 6-well plate. The next day, the growth medium was replaced with RPMI1 hours containing 0.2% BSA (bovine serum albumin). Then 12,5 μ g/mL bispecific antibody was added to the medium and the cells were incubated for 15 minutes, followed by addition of HGF (R & D, 294-HGN) at a final concentration of 25ng/mL and incubation for an additional 10 minutes. Cells were washed once with ice cold PBS containing 1mM sodium vanadate, then placed on ice and lysed in cell culture plates with 100. mu.L lysis buffer (50mM Tris-ClpH7.5, 150mM NaCl, 1% NP40, 0.5% DOC, aprotinin, 0.5mM PMSF, 1mM sodium vanadate). The cell lysate was transferred to eppendorf centrifuge tubes and lysis was allowed to proceed on ice for 30 minutes. Protein concentration was determined using BCA method (Pierce). 30-50. mu.g of lysate were separated on a 4-12% Bis-Tris NuPage gel (Invitrogen) and the proteins on the gel were transferred to nitrocellulose. Membranes were blocked with TBS-T containing 5% BSA for 1 hour and visualized with a phospho-specific c-Met antibody against Y1349 (Epitomics, 2319-1) according to the manufacturer's instructions. The immunoblot was again probed with an antibody that binds non-phosphorylated c-Met (Santa Cruz, sc-161).

Her3(ErbB3) phosphorylation assay

Each well of a 12-well plate was seeded with 2x10e5 MCF7 cells in complete growth medium (RPMI1640, 10% FCS). Cells were allowed to grow to 90% confluence within 2 days. The medium was then replaced with starvation medium containing 0.5% FCS. The next day, supplemented with the indicated concentrations of each antibody, and 1 hour later 500 ng/mL heregulin (R & D) was added. Cells were cultured for an additional 10 minutes after addition of the heregulin, and then harvested and lysed. Protein concentration was determined using BCA method (Pierce). 30-50. mu.g of lysate were separated on a 4-12% Bis-Tris NuPage gel (Invitrogen) and the proteins on the gel were transferred to nitrocellulose. Membranes were blocked with TBS-T containing 5% BSA for 1 hour and visualized with phospho-specific Her 3/ErbB 3 antibody (4791, CellSignaling) that specifically recognizes Tyr 1289.

FACS

A549 was isolated and counted. Each well of the conical 96-well plate was seeded with 1.5 × 10e5 cells. Cells were centrifuged (1500rpm, 4 ℃,5 min) and incubated for 30 min on ice in 50 μ L dilution series of each bispecific antibody in PBS containing 2% FCS (fetal calf serum). The cells were again centrifuged and washed once with 200uL of PBS containing 2% FCS, followed by re-incubation of the cells with 5 μ g/mL of Alexa 488-conjugated antibody against human Fc diluted in PBS containing 2% FCS (Jackson1 mmonesearch, 109116098) for 30 min. Cells were washed 2 times by centrifugation with 200 μ L PBS containing 2% FCS, resuspended in BD CellFix solution (BD Biosciences) and incubated on ice for at least 10 minutes. The mean fluorescence intensity (mfi) of the cells was determined by flow cytometry (FACS Canto, BD). Mfi was determined by at least two independent staining in duplicate. Flow cytometry spectra were further processed using FlowJo software (TreeStar). Half maximal binding was determined using xlfitt 4.0(IDBS) and dose response one site model 205.

Surface plasmon resonance

The binding performance of monovalent anti-IGF-IR antibodies was analyzed by Surface Plasmon Resonance (SPR) techniques using a Biacore instrument (Biacore, GE-Healthcare, Uppsala). This system is well established for studying molecular interactions. It allows continuous real-time monitoring of ligand/analyte binding and thus determination of association rate constants (ka), dissociation rate constants (KD), and equilibrium constants (KD) in a variety of assay settings. SPR technology is based on measuring the refractive index near the surface of a gold-coated biosensor chip. The change in refractive index indicates a mass change on the surface caused by the interaction of the immobilized ligand with the injected analyte in solution. The mass increases if the molecule binds to the ligand immobilized on the surface and decreases in the case of dissociation. For capture, anti-human IgG antibodies were immobilized on the surface of CM5 biosensor chip using amine coupling chemistry. The mixture was purified at a flow rate of 5. mu.l/min using 0.1M N-hydroxysuccinimide and 0.1M3- (N, N-dimethylamino) propyl-N-ethylcarbodiimide 1: 1 the mixture activates the flow cell. Anti-human IgG antibody was injected at 10. mu.g/ml into sodium acetate, pH 5.0. The reference control flow cell was treated in the same manner, but only the carrier buffer was used in place of the capture antibody. The surface was blocked by injection of 1M ethanolamine/HC1pH8.5. IGF-1R antibody was diluted in HBS-P and injected. All interactions were performed at 25 ℃ (standard temperature). After each binding cycle a 60 second 3M magnesium chloride regeneration solution was injected at a flow rate of 5 μ l/min to remove any non-covalently bound proteins. The signals were detected at a rate of 1 signal per second. Samples with increasing concentrations were injected. FIG. 17 depicts the assay format of the application. The capture antibody density and IGF-1R antibody were chosen at low loading density to force monovalent binding.

For affinity measurements, human fcgiia was immobilized on a CM-5 sensor chip by capturing His-tagged receptors with anti-His antibodies (Penta-His, Qiagen) coupled to the surface via standard amine coupling and blocking chemistry on the surface of the SPR instrument (Biacore T100). After FcgRIIIa capture, 50nM IGF1R antibody was injected at 25 ℃ at a flow rate of 5 μ L/min. The chip was then regenerated with a 60 second pulse of 10mM glycine-HC 1, pH2.0 solution.

Antibody-dependent cellular cytotoxicity Assay (ADCC)

Determining effector function mediated by the antibody against IGF-IR antibody. To determine the ability of the antibodies produced to elicit immune effector mechanisms, antibody-dependent cellular cytotoxicity (ADCC) studies were performed. To study the effect of the antibodies in ADCC, DU145IGF-IR expressing cells (1X 10) were labeled with 1. mu.l/ml of BATDA solution (Perkin Elmer) in a cell culture incubator at 37 ℃⁶Individual cells/ml) for 25 minutes. Thereafter, the cells were washed 4 times with 10ml RPMI-FM/PenStrep and centrifuged at 200x g for 10 minutes. Before the last centrifugation step, the cell number was determined and diluted to 1 × 10e5 cells/ml in RPMI-FM/PenStrep medium from the following pellet. A50. mu.l volume of 5,000 cells was placed in each well of the round bottom plate. HuMAb antibody was added to 50. mu.l of the cell suspension at a final concentration in the range 25-0.1. mu.g/ml in a volume of 50. mu.l of cell culture medium. After that, the ratio of 25: 1E: t ratio 50. mu.l of effector cells, freshly isolated PBMC were added. The plates were centrifuged at 200x g for 1 minute followed by a step of incubation at 37 ℃ for 2 hours. After incubation, cells were centrifuged at 200x g for 10 minutes, and 20 μ l of supernatant was harvested and transferred to Optiplate96-F plates. Add 200. mu.l europium solution (Perkin Elmer, at room temperature) and incubate the plate on a shaking table for 15 min. Fluorescence was quantified in time-resolved fluorogens (Victor3, Perkin Elmer) using the Eu-TDA protocol of Perkin Elmer. Expression of% of maximal release of TDA fluorescence enhancer in detergent lysed target cells corrected for spontaneous release of TDA for individual target cellsThe extent of cell lysis by ADCC.

IGF-1R internalization assay

Binding of the antibodies and antigen binding proteins according to the invention to IGF-1R results in internalization and degradation of the receptor. This process can be monitored by incubating IGF-1R-expressing HT29CRC cells with IGF-1R targeting antibodies, followed by quantitation of the level of IGF-1R protein remaining in the cell lysates by ELISA.

For this purpose, 1, 5x10 was incubated overnight in RPMI with 10% FCS in 96-well MTP at 37 ℃ and 5% CO2⁴Cells/wells of HT29 cells to allow cell attachment. The next morning, the medium was aspirated off and added at a concentration of from 10nM to 2pM in a 1: dilution step 3 mu.l of anti-IGF-1R antibody diluted in RPMI + 10% FCS. Cells were incubated with antibody at 37 ℃ for 18 hours. After that, the medium was removed again and 120. mu.l MES lysis buffer (25mM MES pH6.5+ complete) was added.

For ELISA, 100. mu.l of a 96-well streptavidin-coated polystyrene plate (Nunc) was added at a rate of 1: 200 MAK < huIGF-1R α > hu-1a-IgG-Bi (Ch.10) diluted in 3% BSA/PBST (final concentration 2.4. mu.g/m 1) and incubated at room temperature for 1 hour with constant shaking. The well contents were then removed and washed three times with 200 μ l PBST per well. Add 100. mu.l of cell lysis solution to each well, incubate again for 1 hour at room temperature on a plate shaker, and wash three times with 200. mu.l PBST. After removing the supernatant, 100 μ l/well of a suspension of 1: PAK < human IGF-1R α > Ra-C20-IgG (Santa Cruz # sc-713) diluted 750 in 3% BSA/PBST followed by the same incubation and wash intervals as above. To detect specific antibodies that bind IGF-1R, 100. mu.l/well of a 1: polyclonal horse radish peroxidase-conjugated rabbit antibody (Cell Signaling #7074) diluted 4000 in 3% BSA/PBST. After another 1 hour, unbound antibody was removed again by washing 6 times thoroughly as described above. To quantify the bound antibody, 100. mu.l/well of 3,3'-5,5' -tetramethylbenzidine (Roche, BM-Blue ID. -Nr.11484281) was added and incubated at room temperature for 30 minutes. The color reaction was finally stopped by adding 25. mu.l/well of 1M H2SO4 and the light absorption was measured at a wavelength of 450 nm. Cells not treated with antibody were used as a 0% down-regulated control and lysis buffer was used as a background control.

IGF-1R autophosphorylation assay (IGF-1 stimulation)

Targeting of IGF-1R antibodies to IGF-1R results in the inhibition of IGF-1-induced autophosphorylation. We investigated the autophosphorylation inhibition of monovalent IGF-1R antibodies without a binding pore compared to the parent IGF-1R IgG1 antibody. For this purpose, murine fibroblast cell line 3T3-IGF-1R cells overexpressing human IGF-1R were treated with 10nM recombinant human IGF-1 for 10 minutes in the presence of varying concentrations of monovalent and divalent IGF-1R antibodies. After cell lysis, the level of phosphorylated IGF-1R protein was determined by phospho-IGF-1R specific ELISA in combination with a human IGF-1R specific capture antibody and a phospho-tyrosine specific detection antibody.

Measurement of PK Performance: single dose kinetics in mice

Method of producing a composite material

Animals:

NMRI mice, female, housed, weighed 23-32g at the time of compound administration.

The research scheme is as follows:

for a single intravenous dose of 10 mg/kg, mice were divided into 3 groups of 2-3 animals each. Blood samples were taken from group 1 at 0.5, 168 and 672 hours post-dose, from group 2 at 24 and 336 hours post-dose, and from group 3 at 48 and 504 hours post-dose.

Approximately 100 μ L of blood sample was obtained by retrobulbar puncture. At least 40. mu.l of serum sample was obtained from the blood by centrifugation (9300Xg) for 2.5 minutes after 1 hour at room temperature. Serum samples were frozen directly after centrifugation and stored frozen at-20 ℃ until analysis.

And (3) analysis:

the concentration of human antibodies in mouse sera was determined by enzyme-linked immunosorbent assay (ELISA) using 1% mouse sera. In a first step, the person is targetedBiotinylated monoclonal antibodies (mAbs) to Fc gamma<hFcγ_ＰANIgG-Bi) bound to streptavidin coated microtiter plates. In the next step, serum samples (at different dilutions) and reference standards were added separately and allowed to react with the immobilized mAb < hFc γ_ＰANIgG-Bi binding. Then adding digoxin monoclonal antibody (mAb < hFc gamma) aiming at human Fc gamma_ＰAN> IgG-Dig). Human antibodies were detected by anti-Dig-horseradish peroxidase antibody conjugate. ABTS solution was used as substrate for horseradish peroxidase. The specificity of the capture and detection antibodies used, which do not cross-react with mouse IgG, enables the quantitative determination of human antibodies in mouse serum samples.

And (3) calculating:

pharmacokinetic parameters were calculated by non-compartmental (non-comparative) analysis using the pharmacokinetic evaluation program WinNonlin^TMVersion 5.2.1.

Table 1: calculated pharmacokinetic parameters:

human antibodies were evaluated using the following pharmacokinetic parameters:

estimated initial concentration for the bolus IV model (C0).

Maximum observed concentration (C)_max) In (T)_max) And occurs.

Time of maximum observed concentration (T)_max).

The area under the concentration/time curve, AUC (0-inf), calculated by the linear trapezoidal rule (using linear interpolation) from time 0 to infinity.

Apparent terminal half-life (T)_1／2) From the equation: t is_l／2=ln2／λz。

Total Clearance (CL), calculated as dose/AUC (0-inf).

Steady state volume of distribution (Vss), calculated as MRT (0-inf) x CL (MRT (0-inf), defined as AUMC (0-inf)/AUC (0-inf).

B. Example (b):

example 1:

generation of monovalent antibodies

Based on the design principle shown in FIG. 1A, we designed monovalent antigen binding proteins against c-Met (SEQ ID NO: 1 and SEQ ID NO: 2; c-Met5D5MoAb ("wt")), IGF-1R (SEQ ID NO: 3 and SEQ ID NO: 4.; IGFlR AKl8MoAb ("wt")) and HER3(SEQ ID NO: 5 and SEQ ID NO: 6; Her3205MoAb ("wt")). In addition, identical monovalent antibodies directed against c-Met (SEQ ID NO: 7 and SEQ ID NO: 8; c-Met5D5MoAb KiH), IGF-1R (SEQ ID NO: 9 and SEQ ID NO: 10; IGFlR AK18MoAb KiH) and HER3(SEQ ID NO: 11 and SEQ ID NO: 12; Her3205MoAb KiH) were designed which incorporate a mutation in the CH3 portion to support passage through the binding well (A) (SEQ ID NO: 7 and SEQ ID NO: 8)knob-into-hole) (, KiH) technology (Merchant, A.M., et al, nat. Biotechno1.16(1998) 677-. As described above, all monovalent antibodies were transiently expressed in HEK293 cells and subsequently purified by protein a affinity chromatography followed by size exclusion.

FIGS. 3-5 depict a chromatogram of size exclusion chromatography of 3 different monovalent antigen binding proteins that did not bind to the well, and corresponding SDS-PAGE under non-reducing and reducing conditions.

The size of the different peaks was confirmed by SEC-MALLS (fig. 4C) and the identity of the isolated protein was confirmed by mass spectrometry. Collectively, these data show that CHI-CL crossover allows for easy purification of pure monovalent antibodies (peak 3 in fig. 3, peak 2 in fig. 4, peak 3 in fig. 5) without including a binding pore in the Fc portion to force heterodimerization. This product can be separated by size exclusion chromatography from the bivalent, dimeric form of the antigen binding protein (, MoAb dimer) baseline as a by-product, depicted in fig. 1C, prior to the monovalent antigen binding protein peak. Most of the cysteine bridges of the cross-linked dimers in the bivalent, dimeric construct were not closed, which resulted in the observation of the major product observed at 100kDa in SDS-PAGE under non-reducing conditions, rather than at 200kDa as expected (peak 2 in fig. 3, peak 1 in fig. 4, peak 2 in fig. 5). The additional peaks observed for c-Met5D5MoAb ("wt") and Her3205MoAb ("wt") (peak 1 in fig. 3, peak 1 in fig. 5) depict higher molecular weight aggregates. This is in contrast to the monovalent antibodies described in WO/2007/048037, where a mixture of heterodimeric and homodimeric monovalent antibodies could not be isolated by conventional means (FIG. 2).

FIGS. 6-5 depict a chromatogram of size exclusion chromatography of 3 different monovalent antigen binding proteins with binding pores, and corresponding SDS-PAGE under non-reducing and reducing conditions.

By applying this binding-pore technique for Fc heterodimerization, the relative yield of heterodimeric monovalent antigen binding proteins compared to bivalent MoAb dimers can be increased, as shown in fig. 6-8.

Example 2:

c-Met phosphorylation (FIG. 9)

c-Met has been described as an oncogenic tyrosine kinase, whose deregulation facilitates cellular transformation. Antibodies targeting c-Met have been described previously. MetMAb/OA-5D 5(Genentech) is one such antibody that inhibits ligand-dependent activation of c-Met. Because the bivalent antibody is activated, it is engineered as a one-armed construct, in which one FAb arm is deleted, leaving a monovalent antibody. To demonstrate similar potency of OA-5D5 and the monovalent antigen binding protein c-Met MoAb (c-Met5D5MoAb ("wt")), a549 cells and individual antibodies were incubated in the absence or presence of the only known c-Met ligand, HGF. Unlike bivalent MetMAb (biv. ab), none of the antibodies have activation potential in the absence of HGF. Furthermore, as expected, c-Met MoAb (c-Met5D5MoAb ("wt")) was as effective as OA-5D5 in inhibiting ligand-induced receptor phosphorylation. Non-specific human IgG control antibodies had no effect on HGF-dependent c-Met receptor phosphorylation.

Example 3:

binding to cells of c-Met expressing cell lines (FIG. 10)

Cell binding of the monovalent antigen binding protein c-Met MoAb (c-Met5D5MoAb ("wt")) was demonstrated on a549 cells. The cell suspension was incubated with a 3-fold dilution series (100-0.0003. mu.g/mL) of the indicated antibody. Bound antibodies were visualized using Alexa 488-conjugated secondary antibodies that bound to the human immunoglobulin constant region. Single cell fluorescence intensity was measured on a FACS Canto (BD Biosciences) flow cytometer. No difference was observed in the binding of c-Met MoAb and OA-5D5, suggesting that c-Met MoAb (c-Met5D5MoAb ("wt")) binds cell surface c-Met efficiently.

Half maximal binding

OA-5D5： 1.45nM

c-Met MoAb 1.57nM

Example 4:

IGF-1R binding affinity (FIG. 11)

Binding of the monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) and the parent < IGF-1R > IgG1 antibody to the IGF-1R extracellular domain was compared by Surface Plasmon Resonance (SPR). FIG. 17 depicts an SPR assay protocol for determining monovalent affinity. Analysis (2 assays) showed that IGF-1R binding affinity was retained in monovalent antibodies.

Example 5:

binding to cells of IGF-1R expressing cell line (FIG. 12)

Cell binding of the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) was demonstrated on a549 cells. A549 cells in logarithmic growth phase were isolated using accutase (sigma) and 2x10e5 cells were used for incubation of each individual antibody. MoAb was added in a three-fold dilution series (100-0.0003. mu.g/mL). Bound antibodies were visualized using an Alexa 488-conjugated secondary antibody (5 μ g/mL) that binds to the human immunoglobulin constant region. Dead cells were stained with 7-AAD (BD) and excluded from the analysis. Single cell fluorescence intensity was measured on a FACS Canto (BD Biosciences) flow cytometer. The data show a difference in half maximal binding to cells because the IGF-1R IgG1 antibody can bind IGF-1R on cells with two arms and exhibit avidity effects, whereas monovalent antibodies can bind with only one arm.

Half maximal binding

IGF-1R(150kDa)： 0.76nM

IGF-1R MoAb(100kDa)： 5.65nM

Example 6:

ADCC Induction (FIG. 13)

Effector cell recruitment of antibodies by non-glycoengineered and glycoengineered cancer cells can be measured using donor-derived Peripheral Blood Mononuclear Cells (PBMCs). Lysis of cancer cells is associated with NK cell-mediated cytotoxicity and is proportional to the ability of antibodies to recruit NK cells. In this particular setup, DU145 prostate cancer cells were treated in the absence and presence of each antibody at a 1: a ratio of 25 (DU 145: PBMC) was incubated with PBMC. After 2 hours, cell lysis was measured using the BATDA/europium system as described above. The extent of cell lysis by ADCC is expressed as% of the maximal release of TDA fluorescence enhancer in target cells lysed by detergent corrected for the spontaneous release of TDA of the respective target cells. The data show that the non-sugar engineered monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) induces ADCC at high concentrations better than the non-sugar engineered parent IGF-1R antibody, despite a lower apparent affinity for IGF-1R on the cells. Surprisingly, the non-glycoengineered monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) induced ADCC at high concentrations even better than the glycoengineered parent IGF-1R antibody, which showed a decrease in ADCC assays when trending towards high concentrations. Such monovalent IGF-1R antigen binding proteins (IGF1R AK18MoAb ("wt")) that mediate reduced IGF-1R internalization and enhanced ADCC due to reduced internalization (see below) and double the amount of Fc portion occupying the FcRIIIa receptor on effector cells, may therefore represent a promising approach to target IGF-1R on cancer cells; as a non-glycoengineered antibody or as a glycoengineered antibody.

Example 7:

IGF-1R internalization assay (FIG. 14)

Targeting IGF-1R by a bivalent parent IGF-1R antibody results in internalization of IGF-1R. We investigated the internalization performance of the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")). The data in figure 14 show that internalization of IGF-1R is reduced in both potency and absolute internalization when the monovalent antigen binding protein IGF1R MoAb (IGF1RAK18MoAb ("wt")) is bound.

Targeting IGF-1R on tumor cells by bivalent IGF-1R antibodies results in internalization of IGF-1R and lysosomal degradation. We investigated the internalization performance of the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")). To this end, HT29 colon cancer cells were treated with varying concentrations of monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) and a bivalent parent IGF-1R antibody for 18 hours. After cell lysis, the remaining level of IGF-1R protein was determined by IGF-1R specific ELISA.

The graph in fig. 20 shows that internalization of IGF-1R is reduced in both potency and absolute internalization when a monovalent antigen binding protein, IGF1R MoAb (IGF1R AK18MoAb ("wt")) is bound. Maximum internalization was reduced from 83% (IgG1) to 48% (MoAb), and the concentration required for half-maximal inhibition was increased from 0.027nM (IgG1) to 1.5nM (MoAb).

Example 8:

IGF-1R autophosphorylation (IGF-1 stimulation) (FIG. 15)

Targeting IGF-1R by IGF-1R antibodies results in the inhibition of IGF-1-induced autophosphorylation. We investigated the autophosphorylation inhibition of the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) compared to the parent IGF-1R IgG1 antibody. For this purpose, murine fibroblast cell line 3T3-IGF-1R cells overexpressing human IGF-1R were treated with 10nM recombinant human IGF-1 in the presence of varying concentrations of the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) and the bivalent parent IGF-1R antibody for 10 minutes. Following cell lysis, the level of phosphorylated IGF-1R protein was determined by phospho-IGF-1R specific ELISA combining a human IGF-1R specific capture antibody and a phospho-tyrosine specific detection antibody.

The data in fig. 15 show that the monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) can inhibit IGF-1-induced autophosphorylation, although at higher concentrations due to monovalent binding on the cell (lack of avidity effects due to bivalent binding). The concentration required for half-maximal inhibition increased from 1.44nM (IgG1) to 27.9nM (moab). Since the difference in IC50 values for monovalent and bivalent antibodies in IGF-1R autophosphorylation (19 fold) was slightly less pronounced than IGF-1R downregulation (59 fold), the effect of monovalent binding on the downregulation cannot be explained with only a decrease in affinity for IGF-1R.

Example 9:

stability of IGF-1R monovalent antigen binding proteins (FIG. 16)

The stability of the monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) was investigated by dynamic light scattering as described above. Briefly, the aggregation propensity of the monovalent antigen binding protein IGF1R MoAb was assessed by DLS time course experiments at 40 ℃. Over the 5 day period, no measurable increase in hydrodynamic radius (Rh) (see fig. 10) of the separated monomer fraction could be detected (fig. 22).

Example 10:

determination of PK Properties

The pharmacokinetic performance of the monovalent antibodies according to the invention was determined as described above (in the methods section) in a single dose PK study in NMRI mice, female, bred, weighing 23-32g at the time of compound administration.

PK properties are provided in the table below and suggest that the monovalent antigen binding protein IGF1RMoAb (IGF1R AK18MoAb ("wt")) has improved PK properties compared to the parent < IGF-1R > IgG1 antibody.

Table 2: PK Performance summary

		<IGF-1R>IgG1 antibodies	<IGF1R>MoAb
				C0	μg／mL	81.9	298.32
Cmax	μg／mL	80.7	290.2
				Tmax	h	0.5	0.5
AUC0-inf	h＊μg／mL	9349	20159
				term t1／2	h	106.2	148.9
Cl	mL/min/kg	0.018	0.0083
				Vss	L／kg	0.16	0.082

Example 11:

ESI-MS experiments IGF-1R MoAb (FIGS. 17 and 18)

Monovalent antigen binding protein IGF1R MoAb (IGF1R AK18MoAb ("wt")) was transiently expressed and purified by protein a chromatography and size exclusion chromatography. After preparative SEC, the antibody eluting in 2 separate peaks (peak 1 and peak 2) was collected. The analytical SEC of fraction 2 (peak 2) corresponds to a molecular weight of 100kDa, suggesting a defined monomer. SEC-MALS confirmed the initial SEC results and showed an Mw of 99.5kDa for fraction 2 (monomer). SDS-PAGE analysis of this fraction under denaturing and reducing conditions showed a main band with an apparent molecular weight of 50-60 kDa. Fraction 2 (monomer) showed a major band of MW of about 100kDa under non-reducing conditions.

Fraction 1 ═ 165mL

Fraction 2 ═ 190mL

The ESI-MS spectrum of deglycosylated MoAb from fraction 2 shows a series of peaks corresponding to monomers with a mass of 98151 Da.

Table 3: MS data from non-reducing ESI-MS measurements of fraction 2 are summarized.

Fraction(s) of	Molecular weight, monomer (theoretical 98162Da)
		Fraction 2	98151Da

MS measurements of fraction 2 under reducing conditions showed correct sequence and expression of the construct. MS data from fraction 2 showed approximately equal amounts of two different heavy chains with molecular weights of 47959Da and 50211 Da.

Table 4: MS data from reduction ESI-MS measurements under reducing conditions for fraction 2 are summarized.

Example 12:

production of glycoengineered antigen binding proteins

For the production of glycoengineered antigen binding proteins, HEK-EBNA cells were transfected with 4 plasmids using the calcium phosphate method. 2 plasmids encoding antibody chains, one for fusion GnTIII polypeptide expression (GnT-III expression vector) and one for mannosidase II expression (golgi mannosidase II expression vector), in a ratio of 4: 4: 1: 1. cells were cultured in adherent monolayer cultures using DMEM medium supplemented with 10% FCS in T flasks and transfected when cells were 50-80% confluent. For transfection in T150 flasks, fifteen million cells were seeded 24 hours prior to transfection in 25ml DMEM medium supplemented with FCS (10% V/V final concentration) andplace the cells in the medium with 5% CO₂The atmosphere incubator was at 37 ℃ overnight. For each T150 flask to be transfected, the total plasmid vector DNA, divided into light and heavy chain expression vectors, was aliquoted by mixing 94. mu.g, water to a final volume of 469. mu.l, and 469. mu.l 1M CaCl₂Preparation of DNA in solution, CaCl₂And a solution of water. To this solution was added 938. mu.l 50mM HEPES, 280mM NaCl, 1.5mM Na pH 7.05₂HPO₄The solution was immediately mixed for 10 seconds and allowed to stand at room temperature for 20 seconds. The suspension was diluted with 10ml of DMEM supplemented with 2% FCS and added to T150, replacing the existing medium. Then an additional 13ml of transfection medium was added. At 37 ℃ 5% CO₂Cells were cultured for about 17 to 20 hours, and then the medium was replaced with 25ml of DMEM, 10% FCS. Conditioned media were harvested by centrifugation at 210Xg for 15 minutes about 7 days after media exchange, the solution was sterile filtered (0.22um filter) and sodium azide was added to a final concentration of 0.01% w/v and stored at 4 ℃.

Claims

1. A monovalent antigen binding protein comprising

2. A monovalent antigen binding protein according to claim 1, characterized in that

i) The CH3 domain of one heavy chain is altered,

And

ii) altering the CH3 domain of the other heavy chain,

3. A monovalent antigen binding protein according to claim 2, characterized in that

4. A monovalent antigen binding protein according to claim 3, characterized in that

5. A monovalent antigen binding protein according to claims 1 to 4, characterized in that it is of the human IgG1 isotype.

6. A monovalent antigen binding protein according to claim 1, characterized in that it comprises

a) A modified heavy chain comprising SEQ ID NO: 1; and

b) a modified heavy chain comprising SEQ ID NO: 2; or

a) A modified heavy chain comprising SEQ ID NO: 3; and

b) a modified heavy chain comprising SEQ ID NO: 4; or

a) A modified heavy chain comprising SEQ ID NO: 5; and

b) a modified heavy chain comprising SEQ ID NO: 6; or

a) A modified heavy chain comprising SEQ ID NO: 7; and

b) a modified heavy chain comprising SEQ ID NO: 8; or

a) A modified heavy chain comprising SEQ ID NO: 9; and

b) a modified heavy chain comprising SEQ ID NO: 10; or

a) A modified heavy chain comprising SEQ ID NO: 11; and

b) a modified heavy chain comprising SEQ ID NO: 12.

7. A monovalent antigen binding protein according to claim 1, 2,3, 4 or 6, characterized in that the modified heavy chain of a) and b) is of IgG1 isotype and the antigen binding protein is afucosylated, having an amount of fucose of 80% or less of the total amount of oligosaccharides (sugars) at Asn297 of the human IgG1 isotype.

8. A pharmaceutical composition of a monovalent antigen binding protein according to claims 1 to 7.

9. A pharmaceutical composition comprising a monovalent antigen binding protein according to claims 1-7 and at least one pharmaceutically acceptable excipient.

10. A monovalent antigen binding protein according to claims 1 to 7 for use in the treatment of cancer.

11. Use of a monovalent antigen binding protein according to claims 1 to 7 for the manufacture of a medicament for the treatment of cancer.

12. A method of treating a patient in need of therapy, said method characterized by administering to the patient a therapeutically effective amount of a monovalent antigen binding protein according to claims 1 to 7.

13. Method for preparing a monovalent antigen binding protein according to claims 1 to 7

The method comprises the following steps

a) Transforming a host cell with a vector comprising a nucleic acid molecule encoding a monovalent antigen binding protein according to claims 1 to 7,

c) recovering the monovalent antigen binding protein molecule from the culture.

14. A nucleic acid encoding a monovalent antigen binding protein according to claims 1 to 7.

15. A vector comprising a nucleic acid according to claim 14.

16. A host cell comprising a vector according to claim 15.