HK1170506B - Bispecific antigen binding proteins - Google Patents

Description

Bispecific antigen binding proteins

The present invention relates to bispecific antigen binding proteins, methods for their production, pharmaceutical compositions containing said antibodies, and uses thereof.

Background

Engineered proteins, such as bi-or multispecific antibodies capable of binding two or more antigens, are known in the art. Such multispecific binding proteins may be generated using cell fusion, chemical conjugation, or recombinant DNA techniques.

A wide variety of recombinant multispecific antibody formats have recently been developed, such as tetravalent bispecific antibodies obtained by fusing, for example, an IgG antibody format and a single-chain domain (see, for example, Coloma, M.J. et al, Nature Biotech.15(1997) 159-1234; WO 2001/077342; and Morrison, S.L., Nature Biotech.25(2007) 1233-1234).

Also, several other novel forms have been developed which are capable of binding two or more antigens without any longer preserving the core structure of the antibody (IgA, IgD, IgE, IgG or IgM), such as diabodies, triabodies or tetrabodies, minibodies, several single chain forms (scFv, bis-scFv) (Holliger, P. et al, NatureBiotech 23(2005) 1126-1136; Fischer, N. and Leger, O. Pathiology 74(2007) 3-14; Shen, J. et al, J.Immunol. methods 318(2007) 65-74; Wu, C. et al, NatureBiotech 25(2007) 1290-1297).

All such formats use linkers to fuse the antibody core (IgA, IgD, IgE, IgG or IgM) with another binding protein (e.g. scFv) or to fuse e.g. two Fab fragments or scfvs (Fischer, n. and leger, o., Pathobiology 74(2007) 3-14). While it is clear that linkers have advantages in engineering bispecific antibodies, they can also cause problems in the therapeutic setting. In fact, these foreign peptides can elicit an immune response against the linker itself or the junction between the protein and the linker. Furthermore, the flexible nature of these peptides makes them more susceptible to proteolytic cleavage, potentially leading to poor antibody stability, aggregation and increased immunogenicity. In addition, it may be desirable to retain effector functions, such as, for example, Complement Dependent Cytotoxicity (CDC) or Antibody Dependent Cellular Cytotoxicity (ADCC), which are mediated by maintaining a high degree of similarity to naturally occurring antibodies via the Fc portion.

Thus, ideally, one should aim to develop bispecific antibodies that are very similar in general structure to naturally occurring antibodies (such as IgA, IgD, IgE, IgG or IgM) with minimal deviation from human sequences.

In one approach, bispecific antibodies very similar to native antibodies have been generated using the four-source hybridoma (quadroma) technique (see Milstein, c. and Cuello, a.c., Nature 305(1983) 537) -540), which is based on somatic fusion of two different hybridoma cell lines expressing murine monoclonal antibodies with the desired specificity for bispecific antibodies. Due to the random pairing of two different antibody heavy and light chains within the resulting hybrid-hybridoma (or quadroma) cell line, up to 10 different antibody species were generated, only one of which was the desired functional bispecific antibody. The presence of mismatched byproducts and significantly reduced production yields mean that complicated purification procedures are required (see, e.g., Morrison, S.L., Nature Biotech.25(2007) 1233-1234). In general, the same problem of mismatched byproducts remains when recombinant expression techniques are used.

One approach to circumvent the problem of mismatched byproducts, known as "knob-in-hole," is to modify the contact interface by introducing mutations into the CH3 domain, thereby forcing the pairing of two different antibody heavy chains. On one chain, bulky amino acids are replaced with amino acids with shorter side chains to create "holes". Conversely, an amino acid with a larger side chain was introduced into another CH3 domain to create a "knob". By co-expressing both heavy chains (and two identical light chains, which must be adapted to both heavy chains), high yields of heterodimer formation ("knob-hole") relative to homodimer formation ("hole-hole" or "knob-knob") were observed (Ridgway, J.B., Protein Eng.9(1996) 617-621; and WO 96/027011). The percentage of heterodimers can be further increased by using phage display to reconstruct the interaction surface of the two CH3 domains and introducing disulfide bridges to stabilize the heterodimers (MerchantA. M et al, Nature Biotech 16(1998) 677-681; Atwell, S. et al, J.mol.biol.270(1997) 26-35). Novel methods for the bulge-in-pocket technique are described, for example, in EP 1870459A 1. While this format appears very attractive, no data describing the progression to the clinic is currently available. An important constraint of this strategy is that the light chains of the two parent antibodies must be identical to prevent the formation of mismatched and inactive molecules. As such, this technique is not suitable for recombinant bispecific antibodies against two antigens that are easily developed starting from two antibodies against the first and second antigens, since the heavy chains and/or the same light chains of these antibodies must be optimized.

Another approach to circumvent the problem of mismatched by-products in the preparation of bispecific antibodies is to convert from heterodimers to homodimers by using a full length antibody that specifically binds to a first antigen and has fused to its heavy chain N-terminus two fused Fab fragments that specifically bind to a second antigen, as described, for example, in WO 2001/077342. An important disadvantage of this strategy is the formation of unwanted inactive by-products by mismatch of the light chain of the full-length antibody with the CH1-VH domain of the Fab fragment and by mismatch of the light chain of the Fab fragment with the CH1-VH domain of the full-length antibody.

WO 2006/093794 relates to heterodimeric protein binding compositions. WO 99/37791 describes multipurpose antibody derivatives. Morrison, S, L, et al the J.immunolog, 160(1998)2802-2808 indicate the effect of variable region exchange on IgG functional properties.

Summary of The Invention

The present invention encompasses a bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker at the C or N terminus of the heavy or light chain of a); and is

Wherein in the Fab fragment the following modifications are performed:

i) in the two Fab fragments of a), or in the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other,

ii) in both Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and

the constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

or

The constant domains CL and CH1 replace each other,

iii) in both Fab fragments of a),

the variable domains VL and VH are replaced with each other,

or

The constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and

the constant domains CL and CH1 replace each other,

iv) in both Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and is

In the two Fab fragments of b),

the constant domains CL and CH1 replace each other,

or

v) in the two Fab fragments of a),

the constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are substituted for each other.

Yet another embodiment of the present invention is a method for preparing an antigen binding protein according to the present invention, comprising the steps of:

a) transforming a host cell with a vector comprising a nucleic acid molecule encoding a bispecific antigen binding protein according to the invention,

b) culturing said host cell under conditions that allow synthesis of said antibody molecule; and are

c) Recovering the antibody molecule from the culture.

Yet another embodiment of the invention is a host cell comprising a vector comprising a nucleic acid molecule encoding an antigen binding protein according to the invention.

Yet another embodiment of the invention is a pharmaceutical composition comprising an antigen binding protein according to the invention and at least one pharmaceutically acceptable excipient.

Yet another embodiment of the present invention is a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an antigen binding protein according to the invention is administered to said patient.

In accordance with the present invention, the ratio of a desired bispecific antigen binding protein compared to an undesired by-product can be improved by replacing certain domains in a) the Fab fragment of a full length antibody that specifically binds to a first antigen and/or b) two additional fused Fab fragments. In this way, unwanted mismatches of the light chain to the wrong CH1-VH domain can be reduced.

Detailed Description

The present invention encompasses a bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker to the C or N terminus of the heavy chain of a);

and wherein in said Fab fragment the following modifications are performed:

i) in the two Fab fragments of a), or in the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other,

ii) in both Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and

the constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

or

The constant domains CL and CH1 replace each other,

iii) in both Fab fragments of a),

the variable domains VL and VH are replaced with each other,

or

The constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and

the constant domains CL and CH1 replace each other,

iv) in both Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and is

In the two Fab fragments of b),

the constant domains CL and CH1 replace each other,

or

v) in the two Fab fragments of a),

the constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are substituted for each other.

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

the additional Fab fragment is fused via a peptide linker to the C-terminus of the heavy chain of a), or to the N-terminus of the heavy chain of a).

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

the additional Fab fragment is fused via a peptide linker to the C-terminus of the heavy chain of a).

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

the additional Fab fragment is fused via a peptide linker to the N-terminus of the heavy chain of a).

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

in the Fab fragment, the following modifications were performed:

i) in the two Fab fragments of a), or in the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other.

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

in the Fab fragment, the following modifications were performed:

i) in the two Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other.

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

in the Fab fragment, the following modifications were performed:

i) in the two Fab fragments of a),

the constant domains CL and CH1 replace each other.

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

in the Fab fragment, the following modifications were performed:

i) in the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other.

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

in the Fab fragment, the following modifications were performed:

i) in the two Fab fragments of b),

the constant domains CL and CH1 replace each other.

In accordance with the present invention, the ratio of the desired bispecific antigen binding protein compared to the undesired side products (due to mismatch of the light chain to the wrong CH1-VH domain) can be reduced by replacing certain domains in a) the Fab fragment of a full length antibody specifically binding to the first antigen and/or b) in two additional fused Fab fragments. Mismatches in this context mean i) the light chain of the full-length antibody under a) and the CH1-VH domain of the Fab fragment under b); or ii) binding of the light chain of the Fab fragment under b) to the CH1-VH domain of the full length antibody under a) (see fig. 3), which results in unwanted inactive or non-fully functional by-products.

As used herein, the term "antibody" means a full-length antibody consisting of two antibody heavy chains and two antibody light chains (see fig. 1). The heavy chain of the full-length antibody is composed of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1(CH1), an antibody Hinge Region (HR), an antibody heavy chain constant region 2(CH2), and an antibody heavy chain constant domain 3(CH3) (abbreviated as VH-CH1-HR-CH2-CH3) in the N-terminal to C-terminal direction; and optionally antibody heavy chain constant domain 4(CH4) (in the case of antibodies of subclass IgE). Preferably, the heavy chain of the full length antibody is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. The light chain of a full-length antibody is a polypeptide consisting of an antibody light chain variable domain (VL), and an antibody light chain constant domain (CL), abbreviated as VL-CL, in the N-terminal to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Antibody chains are linked together via inter-polypeptide disulfide bonds between the CL domain and CH1 domain (i.e., between the light and heavy chains) and the hinge region of the full-length antibody heavy chain. Examples of typical full-length antibodies are natural antibodies such as IgG (e.g., IgG1 and IgG2), IgM, IgA, IgD, and IgE). Antibodies according to the invention may be from a single species, e.g. human, or they may be chimeric or humanized antibodies. A full-length antibody according to the invention comprises two antigen-binding sites, each formed by a pair of VH and VL, which both specifically bind to the same (first) antigen. 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 antibody comprises two identical Fab fragments consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain. (see fig. 1 and 2).

An "additional Fab fragment" of an antibody that specifically binds a second antigen (see figure 2) refers to an additional Fab fragment consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain of the second antibody. The additional Fab fragment is fused in its unmodified form (see fig. 3) via the heavy chain portion (CH1 or VH domain) to the C-or N-terminus of the heavy or light chain of the antibody that specifically binds the first antigen.

As used within the present invention, the term "peptide linker" means a peptide having an amino acid sequence, which is preferably of synthetic origin. These peptide linkers according to the invention are used to fuse antigen binding peptides to the C-or N-terminus of full-length and/or modified full-length antibody chains to form bispecific antigen binding proteins according to the invention. Preferably, said peptide linker under c) is a peptide having an amino acid sequence with a length of at least 5 amino acids, preferably with a length of 5 to 100, more preferably 10 to 50 amino acids. In one embodiment, the peptide linker is (GxS) n or (GxS) nGm, wherein G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3, 4, 5, or 6, and m ═ 01, 2 or 3) or (x ═ 4, n ═ 2, 3, 4 or 5 and m ═ 0, 1, 2 or 3), preferably x ═ 4 and n ═ 2 or 3, more preferably wherein x ═ 4 and n ═ 2. In one embodiment, the peptide linker is (G)₄S)₂。

As used herein, the term "binding site" or "antigen binding site" means a 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 is derived from an antibody molecule or fragment thereof (e.g., a Fab fragment). An 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 the desired antigen (i.e., the VH/VL pair) may be derived a) from known antibodies to the antigen or b) from novel antibodies or antibody fragments obtained by re-immunization methods using antigenic proteins or nucleic acids or fragments thereof or the like or by phage display.

The antigen binding site of the antigen binding proteins of the present invention contains 6 Complementarity Determining Regions (CDRs) that contribute to the affinity of the binding site for the antigen 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) is determined by comparison with a compiled database of amino acid sequences in which those regions have been defined according to the variability between sequences.

Antibody specificity refers to the selective recognition of a particular epitope of an antigen by an antibody or antigen binding protein. For example, natural antibodies are monospecific. Bispecific antibodies are antibodies with two different antigen binding specificities. In the case of antibodies with more than one specificity, the recognized epitope may be associated with a single antigen or with more than one antigen.

As used herein, the term "monospecific" antibody or antigen binding protein means an antibody or antigen binding protein having one or more binding sites that each bind to the same epitope of the same antigen.

As used within this application, the term "valency" means the presence of a defined number of binding sites in an antibody molecule. For example, a natural antibody or a full-length antibody according to the invention has two binding sites and is bivalent. The term "tetravalent" means that there are four binding sites in the antigen binding protein. As used herein, the term "tetravalent, bispecific" means an antigen binding protein according to the invention having four antigen binding sites, two of which bind to another antigen (or another epitope of an antigen). The antigen binding proteins of the present invention have four binding sites and are tetravalent.

The full length antibodies of the invention comprise immunoglobulin constant regions of one or more immunoglobulin classes. Immunoglobulin classes include the IgG, IgM, IgA, IgD, and IgE isotypes and, in the case of IgG and IgA, the subtypes thereof. In a preferred embodiment, the full length antibodies of the invention have the constant domain structure of an IgG-type antibody.

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to an antibody preparation or antibody or antigen binding protein molecule 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 derived from a different source or species, which is 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 from the constant regions of the original antibody to generate 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 switch antibodies". Chimeric antibodies are the product of an expressed immunoglobulin gene comprising a DNA segment encoding an immunoglobulin variable region and a DNA segment encoding an immunoglobulin constant region. Methods for generating chimeric antibodies involve conventional recombinant DNA and gene transfection techniques, which are well known in the art. See, e.g., Morrison, S., L., et al, Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855; US 5,202,238 and US 5,204,244.

The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDRs) have been modified to comprise immunoglobulin CDRs of different specificity compared to the specificity of the parent immunoglobulin. 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, Nature 332(1988) 323-327; and Neuberger, M.S. et al, Nature 314(1985)268- & 270. Particularly preferred CDRs correspond to those representing sequences that recognize the antigens noted above for the chimeric antibodies. Other forms of "humanized antibodies" encompassed by the invention are those in which the constant regions have been additionally modified or altered from the constant regions of the original antibody to generate properties in accordance with the invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding.

As used herein, the term "human antibody" 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 are capable of producing a complete repertoire or selection of human antibodies after immunization without endogenous immunoglobulin production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies after antigen challenge (see, e.g., Jakobovits, A. et al, Proc. Natl. Acad. Sci. USA 90(1993) 2551-2555; Jakobovits, A. et al, Nature 362(1993) 255-258; Brueggemann, 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) 381-388; Marks, J.D. et al, J.Mol.biol.222(1991) 581-597). The technique of Cole et al and Boerner et al can also be used to prepare human Monoclonal Antibodies (Cole, S., P., C., et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss (1985) 77-96; and Boerner, P., et al, J.Immunol.147(1991) 86-95). As already mentioned in relation to the chimeric and humanized antibodies according to the invention, the term "human antibody", as used herein, also comprises antibodies which are modified in the constant region to generate the properties according to the invention (in particular with respect to C1q binding and/or FcR binding), for example by "class switching", i.e. a change or mutation of the Fc part (for example from IgG1 to IgG4 and/or IgG1/IgG4 mutations).

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, such as antibodies isolated from host cells such as NS0 or CHO cells or from transgenic animals (e.g., mice) of human immunoglobulin genes or antibodies expressed using recombinant expression vectors transfected into host cells. Such recombinant human antibodies have rearranged forms of variable and constant regions. Recombinant human antibodies according to the invention have been subject to somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of the recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur 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 pair of light and heavy chains directly involved in antibody-to-antigen binding. The human light and heavy chain variable domains have the same general structure, and each domain comprises 4 sequence-wide conserved Framework (FR) regions connected by 3 "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation, and the CDRs can form loops connecting the β -sheet structure. The CDRs in each chain retain their three-dimensional structure through the framework regions and form together with the CDRs from the other chain an 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 thus provide a further object of the invention.

The term "hypervariable region" or "antigen-binding portion of an antibody" as used herein 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". The "framework" or "FR" regions are those variable domain regions which differ from the hypervariable region residues as defined herein. Thus, the light and heavy chains of the antibody comprise the N-to C-terminal domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. The CDRs on each chain are separated by such framework amino acids. In particular, CDR3 of the heavy chain is the region most contributing to antigen binding. CDR and FR regions were determined according to the standard definition of Sequences of proteins of Immunological Interest, 5 th edition, Public Health Service, national institutes of Health, Bethesda, MD (1991), Kabat et al.

As used herein, the term "binding" or "specific binding" refers to the binding of an antibody to an epitope in an in vitro assay, preferably in a plasmon resonance assay (BIAcore, GE-Healthcare Uppsala, Sweden) using a purified wild-type antigen. By the term ka (the binding rate constant of an antibody (or antibody or antigen binding protein) from an antibody/antigen complex), k_D(dissociation constant), and K_D(k_D/ka) to define 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, bispecific antigen binding proteins according to the invention are provided in 10^-8mol/l or less, preferably 10^-9M to 10^-13Binding affinity (K) in mol/l_D) Specifically binding to each antigen for which it is specific.

Binding of the antibody to Fc γ RIII can be investigated by BIAcore assay (GE-Healthcare Uppsala, Sweden). By the term ka (binding rate constant of antibody from antibody/antigen complex), k_D(dissociation constant), and K_D(k_D/ka) to define binding affinity.

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

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

In yet another embodiment, the bispecific 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 the mutations L234A and L235A.

In yet another embodiment, the bispecific antigen binding protein according to the invention is characterized in that the full length antibody is of the human IgG2 subclass.

In yet another embodiment, the bispecific antigen binding protein according to the invention is characterized in that the full length antibody is of the human IgG3 subclass.

In yet another embodiment, the bispecific antigen binding protein according to the invention is characterized in that the full length antibody is of the subclass human IgG4, or human IgG4 with the additional mutation S228P.

Preferably, the bispecific antigen binding protein according to the invention is characterized in that the full length antibody is of the subclass human IgG1, human IgG4 with the additional mutation S228P.

It has now been found that bispecific antigen binding proteins according to the invention have improved characteristics, such as biological or pharmacological activity, pharmacokinetic properties or toxicity. They may be used, for example, in the treatment of diseases such as cancer.

As used within this application, the term "constant region" means the sum of the domains in an antibody that differ from the variable region. The constant region is not directly involved in antigen binding, but exhibits multiple effector functions. Antibodies are classified according to the amino acid sequence of their heavy chain constant region into categories: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses, such as IgG1, IgG2, IgG3, and IgG4, IgA1, and IgA 2. The heavy chain constant regions corresponding to different classes of antibodies are referred to as α, δ, ε, γ, and μ, respectively. Light chain constant regions (CL) that can be found in all 5 antibody classes are called kappa (kappa) and lambda (lambda).

As used herein, the term "constant region derived from human origin" means the heavy chain constant region and/or the light chain kappa or lambda constant region of a human antibody of subclass IgG1, IgG2, IgG3, or IgG 4. Such constant regions are well known in the art and are described, for example, by 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 (FcgRIIIa) binding, antibodies of other IgG subclasses showed strong binding. Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, and His435 are residues which when altered also provide reduced Fc receptor binding (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, Immunology 86(1995) 319-324; EP 0307434).

In one embodiment, the antigen binding protein according to the invention has reduced FcR binding compared to the IgG1 antibody, whereas the full length parent antibody is of the subclass IgG4 or of the subclass IgG1 or IgG2 with mutations in S228, L234, L235 and/or D265 with respect to FcR binding and/or contains 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 in IgG4S228P and in IgG1L234A and L235A.

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. Binding of C1q to antibodies is caused by defined protein-protein interactions at the so-called binding site. Such constant region binding sites are known in the art and are described, for example, by Lukas, T.J., et al, J.Immunol.127(1981) 2555-2560; brunhouse, r. and Cebra, j., mol. immunol.16(1979) 907-; burton, D.R. et al, Nature 288(1980) 338-344; thommesen, J., E., et al, mol. Immunol.37(2000) 995-; idusogene, E. et al, J.Immunol.164(2000) 4178-; hezareh, M.et al, J.Virol.75(2001) 12161-; morgan, A. et al, Immunology 86(1995) 319-324; and EP 0307434. Such constant region binding sites are for example characterized by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat).

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

The term "Complement Dependent Cytotoxicity (CDC)" means a process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. Binding of C1q to antibodies is caused by defined protein-protein interactions at the so-called binding site. Such Fc moiety binding sites are known in the art (see above). Such Fc moiety 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). Antibodies of subclasses IgG1, IgG2, and IgG3 are generally shown to include complement activation with C1q and C3 binding, whereas IgG4 does not activate the complement system and 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. IgG 1-type antibodies (the most commonly used therapeutic antibodies) are glycoproteins with a conserved N-linked glycosylation site at Asn297 in each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are buried between CH2 domains, which form extensive contacts with the polypeptide backbone, and their presence is critical for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, m., r. et al, Glycobiology 5(1995) 813-822; Jefferis, r. et al, immunol. rev.163(1998) 59-76; Wright, a. and Morrison, s., l., Trends biotechnol.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 bisected oligosaccharides) in Chinese Hamster Ovary (CHO) cells significantly increases the ADCC activity of the antibody in vitro. Modification of the composition of Asn297 carbohydrate or its elimination also affects binding to Fc γ R and C1q (Umana, P. et al, Nature Biotechnol.17(1999) 176-180; Davies, J. et al, Biotechnol.Bioeng.74(2001) 288-294; Mimura, Y. et al, J.biol.Chem.276(2001) 45539-45547; Radaev, S. et al, J.biol.Chem.276(2001) 16478-16483; Shields, R.L.et al, J.biol.Chem.276(2001) 6591-6604; Shields, R.L.et al, J.Chem.277 (2002) 26740; Simmons, L.C. et al, J.Biomunol.263) 147).

Methods for enhancing cell-mediated effector function of monoclonal antibodies are reported, for example, in WO 2005/018572, WO 2006/116260, WO 2006/114700, WO2004/065540, WO 2005/011735, WO 2005/027966, WO 1997/028267, US2006/0134709, US 2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739.

In a preferred embodiment of the invention, the bispecific antigen binding protein is glycosylated with a sugar chain at Asn297 (if it comprises an Fc moiety of the subclass IgG1, IgG2, IgG3 or IgG4, preferably of the subclass IgG1 or IgG 3), wherein the amount of fucose within said sugar chain is 65% or less (according to Kabat numbering). In another embodiment, the amount of fucose within said sugar chain is between 5% and 65%, preferably between 20% and 40%. "Asn 297" according to the invention means the amino acid asparagine located at about position 297 in the Fc region. Asn297 may also be located some amino acids (usually no more than ± 3 amino acids) upstream or downstream of position 297, i.e. between positions 294 and 300, based on minor sequence variations of the antibody. In one embodiment, the glycosylated antigen binding protein IgG subclass according to the invention is of the human IgG1 subclass, of the human IgG1 subclass with mutations L234A and L235A, or of the IgG3 subclass. In yet another embodiment, the amount of N-glycolylneuraminic acid (NGNA) in the sugar chain is 1% or less and/or the amount of N-terminal alpha-1, 3-galactose is 1% or less. Preferably, the sugar chain exhibits the characteristics of an N-linked glycan attached to Asn297 of an antibody recombinantly expressed in CHO cells.

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

As used within this application, the term "NGNA" means the sugar residue N-glycolylneuraminic acid.

Glycosylation of human IgG1 or IgG3 occurs at Asn297, with the bi-antennary complex oligosaccharide being glycosylated as a core with up to two Gal residues as termini. Kabat, E.E., A. et al, Sequences of proteins of Immunological Interest, published Health Service 5, National Institutes of Health, Bethesda, MD. (1991) and Brueggemann, M. et al, J.exp.Med.166(1987) 1351-; love, T., W, et al, Methods enzymol.178(1989) 515-. Depending on the amount of terminal Gal residues, these structures are designated G0, G1 (. alpha. -1, 6-or. alpha. -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, by Router, F.H., Glycoconjugate J.14(1997) 201-207. Antibodies recombinantly expressed in CHO host cells that are not sugar modified typically undergo fucosylation at Asn297 in an amount of at least 85%. The modified oligosaccharides of the full-length parent antibody may be hybrid or complex. Preferably, the bisected, reduced/nonfucosylated oligosaccharides are hybrid. In another embodiment, the bisected, reduced/nonfucosylated oligosaccharides are complex.

According to the present invention, "amount of fucose" means an amount of the sugar within the sugar chain at Asn297, measured by MALDI-TOF mass spectrometry, and calculated as an average value, relative to the sum of all sugar structures (e.g., complex, hybrid and high mannose structures) attached to Asn 297. The relative amount of fucose is the percentage of fucose-containing structures relative to all sugar structures identified in the N-glycosidase F-treated sample (e.g. complexed, heterozygous and oligo and high mannose structures, respectively) by MALDI-TOF.

The antigen binding proteins according to the invention are produced by recombinant means. Thus, one aspect of the invention is a nucleic acid encoding an antigen binding protein according to the invention, and yet another aspect is a cell comprising said nucleic acid encoding an antigen binding protein according to the invention. Methods for recombinant production are generally known in the art and include protein expression in prokaryotic and eukaryotic cells, followed by isolation of the antigen binding protein and usually purification to a pharmaceutically acceptable purity. For expression of the antibody in a host cell as described above, the nucleic acids encoding each of the modified light and heavy chains are inserted into an expression vector 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 antigen binding protein 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 review articles 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) 151-160; werner, R.G., Drug Res.48(1998) 870-.

Bispecific antigen binding proteins according to the invention are suitably isolated from the culture broth by conventional immunoglobulin purification procedures, such as, for example, protein a-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA or RNA encoding the monoclonal antibody is readily isolated and sequenced using conventional procedures. Hybridoma cells can serve as a source of such DNA and RNA. Once isolated, the DNA may be inserted into an expression vector, which is then transfected into a host cell such as an HEK293 cell, CHO cell, or myeloma cell that does not otherwise produce immunoglobulin protein, to obtain synthesis of the recombinant monoclonal antibody in the host cell.

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

As used herein, the term "host cell" means any kind of cellular 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. As such, the words "transformant" and "transformed cell" include the primary subject cell and cultures derived therefrom, regardless of the number of deliveries. 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 screened for in the originally transformed cell are included.

For example, Barnes, L.M., et al, Cytotechnology 32(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.USA 86(1989) 3833-3837; carter, P.et al, Proc.Natl.Acad.Sci.USA89(1992) 4285-; and Norderhaug, l, et al, j.immunol.methods 204(1997)77-87, describe the cloning of variable domains. A preferred transient expression system (HEK 293) is described by Schlaeger, E.J. and Christensen, K., in Cytotechnology 30(1999)71-83 and Schlaeger, E.J., in J.Immunol. methods 194(1996) 191-199.

Suitable control sequences for prokaryotes include, for example, promoters, optionally operator sequences, and ribosome binding sites. Eukaryotic cells are known to utilize promoters, enhancers, and polyadenylation signals.

A nucleic acid is "operably linked" when it is placed in a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or operably linked to a coding sequence if the ribosome binding site is positioned so as to facilitate translation. In general, "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 achieved by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Antibody purification to eliminate cellular components or other contaminants, such as other cellular nucleic acids or proteins, 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, Current Protocols in molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widely used for protein purification, such as affinity chromatography with 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., with β -mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g., with phenyl-Sepharose, azaarene resin (aza-arenophilic resin), or m-aminophenylboronic acid), metal chelate affinity chromatography (e.g., with ni (ii) -and cu (ii) -affinity materials), size exclusion chromatography, and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (Vijayalakshmi, m.a., appl.biochem.biotech.75(1998) 93-102).

One aspect of the invention is a pharmaceutical composition comprising an antigen binding protein according to the invention. Another aspect of the invention is the use of an antigen binding protein according to the invention for the preparation of a pharmaceutical composition. Yet another aspect of the invention is a method for preparing a pharmaceutical composition comprising an antigen binding protein according to the invention. In another aspect, the invention provides a composition, e.g., a pharmaceutical composition, comprising an antigen binding protein according to the invention formulated with a pharmaceutically acceptable carrier.

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

Another aspect of the invention is a bispecific antigen binding protein according to the invention for use in the treatment of cancer.

Another aspect of the invention is the use of an antigen binding protein according to the invention for the preparation 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 to said patient in need of such treatment an antigen binding protein according to the invention.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coating materials, antibacterial and antifungal agents, isotonic and absorption delaying 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 may be administered by a variety of methods known in the art. As the skilled artisan will appreciate, the route and/or pattern of administration will vary with the desired result. In order to administer a compound of the invention by certain routes of administration, it may be necessary to coat the compound with a material or to co-administer the compound with a material to prevent its inactivation. For example, the compounds can be administered to a subject in a suitable carrier, such as 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 in situ preparation of sterile injectable solutions or dispersions. The use of such media and agents for pharmaceutically active substances is known in the art.

As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is 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.

As used herein, the term cancer refers to a proliferative Disease such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer (bronchoolliololalecular cancer), bone cancer, pancreatic cancer, skin cancer, head and neck cancer (cancer of the head or the kidney), skin or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region (cancer of the anal region), stomach cancer (stomach cancer), stomach cancer (gastric cancer), colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes (carcinoma of the villopa tumors), endometrial cancer (carcinoma of the endometrium), cervical cancer (carcinoma of the esophagus), vaginal cancer (carcinoma of the esophagus), vulvar cancer (carcinoma of the parathyroid), thyroid cancer (carcinoma of the thyroid), thyroid cancer, esophageal cancer, thyroid cancer, cervical cancer, thyroid cancer, bladder cancer, adrenal cancer, soft tissue sarcoma, urinary tract cancer, penile cancer, prostate cancer, bladder cancer, kidney or ureter cancer, renal cell carcinoma, renal pelvis cancer (carcinoma of the renal glands), mesothelioma, hepatocellular carcinoma, cholangiocarcinoma (biliary cancer), Central Nervous System (CNS) neoplasms, spinal axis tumors (spinal axis tumors), brain stem gliomas, glioblastoma multiforme (glioblastomas), astrocytomas (astrocytoma), schwanomas (schwanomas), ependymomas (ependomomona), medulloblastomas (medulloblastomas), meningiomas (menigioma), squamous cell carcinoma (squamocell carcinoma), pituitary adenomas (pituitary adenoma), and Ewings's sarcoma, 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 aseptic procedures, see above, and by the inclusion of various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and the like. It may also be desirable to incorporate isotonic agents, such as sugars, sodium chloride, and the like into the composition. In addition, delayed absorption of the injectable pharmaceutical form may be brought about by the inclusion of agents which delay absorption such as 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 so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response, composition, and mode of administration for a particular patient, yet is non-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, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

The composition must be sterile and fluid to the extent that the composition is deliverable 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 coating materials such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it is preferred to include isotonic agents (e.g., sugars, polyols such as mannitol or sorbitol, and sodium chloride) in the composition.

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 the primary subject cell 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 screened for in the originally transformed cell are included. Where a unique name is intended, it may be evident from the context.

As used herein, the term "transformation" refers to the process of transferring a vector/nucleic acid into a host cell. If cells without an intractable cell wall barrier are used as host cells, transfection is carried out, for example, by calcium phosphate precipitation, as described by Graham, F., L. and Van der Eb, A., J., Virology 52(1973) 456-467. However, other methods for introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing rigid cell wall structures are used, one method of transfection is, for example, calcium treatment with calcium chloride, as described by Cohen, S., N.et al, PNAS.69(1972) 2110-2114.

As used herein, "expression" refers to the process of transcribing a nucleic acid into mRNA and/or the subsequent translation of the transcribed mRNA (also called 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 the eukaryotic cell may include splicing of mRNA.

"vector" refers to a nucleic acid molecule, particularly self-replicating, that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily in the insertion of DNA or RNA into a cell (e.g., chromosomal integration), replicating vectors that function primarily in the replication of DNA or RNA, and expression vectors that function in the transcription and/or translation of DNA or RNA. Also included are vectors that provide more than one function, as described.

An "expression vector" refers to a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. An "expression system" generally refers to a suitable host cell containing 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.

Description of the sequence listing

SEQ ID NO: 1 unmodified heavy chain with C-terminal fused modified Fab fragment < VEGF > VH-CL Domain < Ang-2> (CH1-CL exchange)

SEQ ID NO: 2 modified Fab fragment < VEGF > VL-CH1 domain (CH1-CL exchange)

SEQ ID NO: 3 unmodified light chain < Ang-2>

SEQ ID NO: 4 unmodified heavy chain < Ang-2> with N-terminal fused modified Fab fragment < VEGF > VH-CL domain (CH1-CL exchange)

SEQ ID NO: 5 modified heavy chain having a C-terminal fused unmodified Fab fragment < Ang-2> VH-CH1 domain < VEGF > (CH1-CL exchange)

Brief Description of Drawings

Fig. 1 is a schematic structure of a full-length antibody without a CH4 domain that specifically binds to first antigen 1, having two pairs of heavy and light chains comprising variable and constant domains in a typical order.

Fig. 2a and 2b schematic structures of typical unmodified Fab fragments with peptide linkers at the C-terminus (fig. 2a) or N-terminus (fig. 2b) of the CH1-VH chain specifically binding to second antigen 2.

Fig. 3 schematic structure of a full length antibody specifically binding to a first antigen 1 (fig. 3a) having fused to its heavy chain N-terminus two unmodified Fab fragments specifically binding to a second antigen 2 and unwanted side products resulting from mismatches (fig. 3b, and fig. 3 c).

Fig. 4a, 4b and 4c schematic structures of bispecific antigen binding proteins according to the present invention, wherein mismatches are reduced by replacing certain domains in a) a Fab fragment of a full length antibody specifically binding to first antigen 1 and/or b) two additional fused Fab fragment antibodies specifically binding to second antigen 2. Figure 4a shows a bispecific antigen binding protein. Figures 4b and 4c show all combinations of VH/VL and/or CH1/CL domain exchanges within the full-length Fab fragment and additional Fab fragments that result in bispecific antigen binding proteins according to the invention with reduced mismatches.

FIG. 5 schematic structure of a bispecific antigen binding protein according to the invention (example 1) recognizing Ang-2 and VEGF.

FIG. 6 schematic structure of a bispecific antigen binding protein according to the invention (example 2) recognizing Ang-2 and VEGF.

FIG. 7 schematic structure of a bispecific antigen binding protein according to the invention (example 3) recognizing Ang-2 and VEGF.

Examples

Materials and general methods

For general information on the nucleotide sequences of human immunoglobulin light and heavy chains see: kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, Public Health service, National Institutes of Health, Bethesda, Md. (1991). The amino acids of the antibody chain are numbered and referred to according to EU numbering (Edelman, G.M. et al, Proc. Natl.Acad. Sci. USA 63(1969) 78-85; Kabat, E.A. et al, Sequences of Proteins of Immunological Interest, 5 th edition, public Health Service, National Institutes of Health, Bethesda, Md. (1991)).

Recombinant DNA technology

The DNA is manipulated using standard methods, such as those described in Sambrook, j, et al, Molecular cloning: a laboratory manual; cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989. Molecular biological reagents were used according to the manufacturer's instructions.

Gene synthesis

The desired gene segment is prepared from oligonucleotides generated by chemical synthesis. Gene segments flanked by single restriction endonuclease cleavage sites are assembled by annealing and ligation of oligonucleotides, including PCR amplification, followed by cloning into pcrscript (stratagene) -based pGA4 cloning vectors via designated restriction sites, such as KpnI/SacI or AscI/PacI. The DNA sequence of the subcloned gene fragments was confirmed by DNA sequencing. The gene synthesis fragments were ordered according to the given specifications in Geneart (Regensburg, Germany).

DNA sequencing

The DNA sequence was determined by double-strand sequencing carried out in MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany). DNA and protein sequence analysis and sequence data management

Sequence creation, localization, analysis, annotation and instantiation were performed using the GCG (Genetics Computer Group, Madison, Wisconsin) software package version 10.2 and the Vector NT1 Advance suite version 8.0 of Infmax.

Expression vector

For the expression of the described bispecific tetravalent antibodies, variants of the expression plasmids for transient expression (e.g. in HEK293EBNA or HEK293-F cells) are applied which are based on cDNA constructs with or without a CMV-intron a promoter or on genomic constructs with a CMV promoter.

In addition to the antibody expression cassette, the vector contains:

an origin of replication which allows the replication of this plasmid in E.coli, and

-a beta-lactamase gene conferring ampicillin resistance in E.coli.

The transcription unit of the antibody gene is composed of the following elements:

unique restriction sites at the 5' end

Immediate early enhancer and promoter from human cytomegalovirus,

in the case of cDNA construction, followed by an intron A sequence,

-the 5' -untranslated region of a human antibody gene,

an immunoglobulin heavy chain signal sequence,

human bispecific tetravalent antibody chains (wild type or with domain exchange) as cDNA or as genomic construct with immunoglobulin exon-intron construct

-a 3' untranslated region having a polyadenylation signal sequence, and

a unique restriction site at the 3' end.

Fusion genes comprising the described antibody chains as described below are generated by PCR and/or gene synthesis and assembled by known recombinant methods and techniques, for example by ligating compatible nucleic acid segments using unique restriction sites in each vector. The subcloned nucleic acid sequences were confirmed by DNA sequencing. For transient transfection, larger quantities of plasmid (Nucleobond AX, Macherey-Nagel) were prepared by plasmid preparations from transformed E.coli cultures.

Cell culture technique

Standard Cell culture techniques are used, such as those described in Current Protocols in Cell Biology (2000), Bonifacino, j.s., Dasso, m., Harford, j.b., Lippincott-Schwartz, j.and Yamada, K.M (eds.), John Wiley & Sons, Inc.

Bispecific tetravalent antibodies were expressed by transient co-transfection of each of the three expression plasmids in adherently grown HEK293-EBNA or in suspension grown HEK29-F cells, as described below. Transient transfection in the HEK293-EBNA System

Adherently growing HEK293-EBNA cells (human embryonic kidney cell line 293 expressing the Epstein-Barr virus nuclear antigen; American type culture) cultured in DMEM (Dulbecco's modified Eagle's Medium, Gibco) supplemented with 10% ultra-low IgG FCS (fetal calf serum, Gibco), 2mM L-glutamine (Gibco), and 250. mu.g/ml geneticin (Gibco)Deposit No. ATCC # CRL-10852, lot.959218) were transiently co-transfected with each of three expression plasmids (e.g., encoding a modified heavy chain, and corresponding light chain and modified light chain) to express the bispecific tetravalent antibody. For transfection, FuGENE was used^TMThe ratio of reagent (. mu.l) to DNA (. mu.g) was 4: 1 (range 3: 1 to 6: 1) using FuGENE^TM6 transfection reagent (Roche Molecular Biochemicals). Proteins were expressed from each plasmid separately using a molar ratio of (modified and wild-type) light chain and modified heavy chain encoding plasmids of 1: 1 (equimolar), which ranged from 1: 2 to 2: 1. On day 3 with L-glutamine, glucose [ Sigma ] added to 4mM]And NAA [ Gibco ]]Feeder cells. Cell culture supernatants containing bispecific tetravalent antibody were harvested by centrifugation at day 5 to day 11 post transfection and stored at-20 ℃. For general information on recombinant expression of human immunoglobulins in, for example, HEK293 cells, see: meissner, P, et al, Biotechnol.Bioeng.75(2001) 197-203.

Transient transfection in the HEK293-F System

Alternatively, bispecific tetravalent antibodies were generated by transient transfection of individual plasmids (e.g., encoding the modified heavy chain, and the corresponding light chain and modified light chain) using the HEK293-F system (Invitrogen) according to the manufacturer's instructions. Briefly, HEK293-F cells (Invitrogen) grown in suspension in serum-free FreeStyle 293 expression medium (Invitrogen) in shake flasks or in stirred fermentors were transfected with a mixture of three expression plasmids as described above and 293fectin or fectin (Invitrogen). For 2L shake flasks (Corning), HEK293-F cells were seeded at a density of 1.0E 6 cells/mL in 600mL and at 120rpm with 8% CO₂And (4) incubation. The following day, cells were transfected with a cell density of about 1.5E 6 cells/mL using a mixture of about 42mL A)20mL Opti-MEM (Invitrogen) with 600 μ g total plasmid DNA (1 μ g/mL) (encoding the modified heavy chain, corresponding light chain and corresponding modified light chain in an equimolar ratio) and B)20mL Opti-MEM +1.2mL 293Fectin or Fectin (2 μ l/mL). The glucose solution is added during the fermentation process in accordance with the glucose consumption. Harvesting after 5-10 days containing secreted antibodySupernatant and purifying the antibody directly from the supernatant, or freezing and storing the supernatant.

Protein assay

The Protein concentration of the purified bispecific tetravalent antibody and derivative was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence according to Pace, C., N.et al, Protein Science 4(1995) 2411-1423.

Determination of antibody concentration in supernatant

The concentration of bispecific tetravalent antibody in cell culture supernatants was assessed by immunoprecipitation with protein a agarose beads (Roche). 60 μ L of protein A agarose beads were washed three times in TBS-NP40(50mM Tris, pH 7.5, 150mM NaCl, 1% Nonidet-P40). Subsequently, 1-15mL of cell culture supernatant was applied to protein a agarose beads pre-equilibrated in TBS-NP 40. After 1 hour incubation at room temperature, the beads were applied to an Ultrafree-MC-Filter column (Amicon)]The column was washed once with 0.5mL TBS-NP40, twice with 0.5mL 2 Xphosphate buffered saline (2xPBS, Roche), and 4 brief 0 washes with 0.5mL 100mM sodium citrate pH5. By adding 35. mu.lLDS sample buffer (Invitrogen) to elute bound antibody. Half of the sample was separately mixed withThe sample reducing agents were combined or left unreduced and heated at 70 ℃ for 10 minutes. Therefore, 5-30 μ l was applied to 4-12%Bis-Tris SDS-PAGE (Invitrogen) (MOPS buffer for non-reducing SDS-PAGE and MOPS buffer for reducing SDS-PAGE)MES buffer of antioxidant running buffer additive (Invitrogen)) And stained with coomassie blue.

The concentration of bispecific tetravalent antibody in the cell culture supernatant was quantitatively measured by affinity HPLC chromatography. Briefly, cell culture supernatants containing antibodies and derivatives that bind protein A were applied to 200mM KH on an Agilent HPLC 1100 system₂PO₄100mM sodium citrate, Applied Biosystems Poros A/20 column in pH7.4, and eluted from the matrix with 200mM NaCl, 100mM citric acid, pH 2, 5. Eluted protein was quantified by UV absorbance and peak area fractions. Purified standard IgG1 antibody served as standard.

Alternatively, the concentration of bispecific tetravalent antibody in the cell culture supernatant was measured by sandwich-IgG-ELISA. Briefly, StreptaWell high binding streptavidin A-96 well microtiter plates (Roche) were coated with 100. mu.L/well of 0.1. mu.g/mL biotinylated anti-human IgG capture molecule F (ab') 2< h-Fc γ > BI (Dianova), either at room temperature for 1 hour or alternatively overnight at 4 ℃, followed by three washes with 200. mu.L/well PBS, 0.05% Tween (PBST, Sigma). Dilution series of 100 μ L/well cell culture supernatant containing each antibody in pbs (sigma) were added to the wells and incubated on a microtiter plate shaker at room temperature for 1-2 hours. The wells were washed three times with 200 μ L/well PBST and bound antibody was detected on a microtiter plate shaker at room temperature for 1-2 hours with 100 μ L of 0.1 μ g/mL F (ab') 2< hFc γ > POD (Dianova) as the detection antibody. Unbound detection antibody was washed off three times with 200 μ Ι/well PBST and bound detection antibody was detected by adding 100 μ Ι ABTS/well. The absorbance measurements were carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

Protein purification

Proteins were purified from filtered cell culture supernatant according to standard protocols. Briefly, bispecific tetravalent antibody was applied to a protein a Sepharose column (GE healthcare) and washed with PBS. Elution of bispecific tetravalent antibody was achieved at pH 2.8 followed by immediate neutralization of the sample. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20mM histidine, 150mM NaCl pH 6.0. The monomer fractions are combined, concentrated, if necessary, using, for example, a MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator, frozen, and stored at-20 ℃ or-80 ℃. A portion of the sample is provided for subsequent protein analytical and analytical characterization (e.g., by SDS-PAGE, size exclusion chromatography, or mass spectrometry).

SDS-PAGE

According to manufacturer's instructions, usingPre-gel systems (Invitrogen). Specifically, 10% or 4-12% is usedBis-TRIS precast gel (pH 6.4) andMES (reducing gel, havingAntioxidant running buffer additive) or MOPS (non-reducing gel) running buffer.

Size exclusion chromatography for analysis

Size exclusion chromatography for determining the aggregation and oligomerization status of the bispecific tetravalent antibody was performed by HPLC chromatography. Briefly, protein A purified antibody was applied to an Agilent HPLC 1100 system at 300mM NaCl, 50mM KH₂PO₄/K₂HPO₄Tosoh TSKgelG3000SW column at pH 7.5, or Superdex 200 column in 2 × PBS on a Dionex HPLC-system (GE Healthcare). Eluted protein was quantified by UV absorbance and peak area fractions. BioRad gel filtration standards 151-1901 served as standards.

Mass spectrometry

The total deglycosylation mass of the bispecific tetravalent antibody was determined and confirmed via electrospray ionization mass spectrometry (ESI-MS). Briefly, 100. mu.g of purified antibody was applied at 100mMKH with 50mU₂PO₄/K₂HPO₄N-glycosidase F (PNGaseF, Prozyme) in pH7 was deglycosylated at 37 ℃ for 12-24 hours at protein concentrations up to 2mg/ml, followed by desalting via HPLC on a Sephadex G25 column (GEHealthcare). After deglycosylation and reduction, the respective masses of the modified heavy chain, light chain and modified light chain were determined by ESI-MS. Briefly, 50 μ g of bispecific tetravalent antibody in 115 μ l was incubated with 60 μ l of 1M TCEP and 50 μ l of 8M guanidine hydrochloride followed by desalting. The total mass and the mass of the reduced heavy and light chains were determined via ESI-MS on a Q-Star Elite MS system equipped with a NanoMate source.

VEGF binding ELISA

The binding properties of the bispecific tetravalent antibody were evaluated in an ELISA assay using the full length VEGF165-His protein (R & D Systems). For this purpose, Falcon polystyrene clean-enhanced microtiter plates were coated with 100 μ l of 2 μ g/mL recombinant human VEGF165(R & D Systems) in PBS for 2 hours at room temperature or overnight at 4 ℃. The wells were washed three times with 300. mu.l PBST (0, 2% Tween 20) and blocked with 200. mu.l 2% BSA 0, 1% Tween20 for 30 minutes at room temperature, followed by three washes with 300. mu.l PBST. Serial dilutions of 100 μ L/well purified bispecific tetravalent antibody in pbs (sigma) were added to the wells and incubated on a microtiter plate shaker at room temperature for 1 hour. The wells were washed three times with 300 μ L PBST (0, 2% Tween 20) and bound antibody was detected on a microtiter plate shaker at room temperature for 1 hour with 100 μ L/well of 0.1 μ g/ml F (ab') < hFcgamma > pod (immuno research) in 2% BSA 0, 1% Tween20 as detection antibody. Unbound detection antibody was washed off three times with 300 μ Ι/well PBST and bound detection antibody was detected by addition of 100 μ Ι ABTS/well. The absorbance measurements were carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

VEGF binding: kinetic characterization of VEGF binding by surface plasmon resonance (Biacore) at 37 deg.C

To further confirm ELISA findings, the binding of bispecific tetravalent antibodies to VEGF was quantitatively analyzed using surface plasmon resonance techniques on a Biacore T100 instrument according to the following protocol and using the T100 software package: briefly, bispecific tetravalent antibody was captured on a CM 5-chip via binding goat anti-human IgG (JIR 109-005-098). Capture antibodies were immobilized by amino coupling using standard amino coupling as follows: HBS-N buffer served as running buffer, targeting a ligand density of 700 RUs to complete activation by a mixture of EDC/NHS. The capture antibody was diluted in coupling buffer NaAc, ph5.0, c 2 μ g/mL, and finally the still activated carboxyl groups were blocked by injection of 1M ethanolamine. Capture of bispecific tetravalent < VEGF > antibody was done at 5 μ L/min flow and c ═ 10nM (diluted with running buffer +1mg/mL BSA); a capture level of about 30 RUs should be reached. rhVEGF (rhVEGF, R & D-Systems catalog number 293-VE) was used as the analyte. Kinetic characterization of VEGF binding to bispecific tetravalent < VEGF > antibodies was performed in PBS + 0.005% (v/v) Tween20 as running buffer at 25 ℃ or 37 ℃. Samples were injected at a flow of 50 μ L/min with 80 seconds of binding time and 1200 seconds of dissociation time in a rhVEGF concentration series of 300-0.29 nM. Regeneration of the free capture antibody surface was performed after each analyte cycle with 10mM glycine pH 1.5 and 60 seconds contact time. Kinetic constants were calculated by using the usual double reference method (control reference: binding of rhVEGF to the capture molecule goat anti-human IgG, measurement of blank on flow cell, rhVEGF concentration "0", mode: langmuir binding 1: 1, (Rmax set to local due to capture antibody binding)).

Ang-2 binding ELISA

The binding properties of the bispecific tetravalent antibody were evaluated in an ELISA assay with the full-length Ang-2-His protein (R & D Systems). For this purpose Falcon polystyrene clean-enhanced microtiter plates were coated with 100. mu.l of 1. mu.g/mL recombinant human Ang-2(R & D Systems, without vector) in PBS for 2 hours at room temperature or overnight at 4 ℃. The wells were washed three times with 300. mu.l PBST (0, 2% Tween 20) and blocked with 200. mu.l 2% BSA 0, 1% Tween20 for 30 minutes at room temperature, followed by three washes with 300. mu.l PBST. Serial dilutions of 100 μ L/well purified bispecific tetravalent antibody in pbs (sigma) were added to the wells and incubated on a microtiter plate shaker at room temperature for 1 hour. The wells were washed three times with 300 μ L PBST (0, 2% Tween 20) and bound antibody was detected on a microtiter plate shaker at room temperature for 1 hour with 100 μ L/well of 0.1 μ g/ml F (ab') < hk > POD (Biozol catalogue No. 206005) in 2% BSA 0, 1% Tween20 as the detection antibody. Unbound detection antibody was washed off three times with 300 μ Ι/well PBST and bound detection antibody was detected by addition of 100 μ Ι ABTS/well. The absorbance measurements were carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

Ang-2 binding BIACORE

The binding of the bispecific tetravalent antibody to human Ang-2 was investigated by surface plasmon resonance using a BIACORE T100 instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, goat < hIgG-fcy > polyclonal antibody was immobilized on a CM5 chip via amine coupling to present a bispecific tetravalent antibody to human Ang-2. Binding was measured in HBS buffer (HBS-P (10 mh eps, 150mM NaCl, 0.005% Tween20, ph 7.4), 25 ℃. purified Ang-2-His (R & D Systems or internally purified) was added at various concentrations in solution, binding was measured by Ang-2 injection for 3 minutes, dissociation was measured by washing the chip surface with HBS buffer for 3 minutes, and KD values were assessed using the 1: 1 langmuir binding mode. no 1: 1 binding could be observed due to the heterogeneity of Ang-2 preparations, as such, KD values were only relative estimates. Qiagen No.34660) captured Ang-2 at a capture level of 2000-1700 RU, the pentahis antibody was immobilized on a CM5 chip via amine coupling (no BSA) (see below).

Ang-2-VEGF bridging ELISA

The binding properties of the bispecific tetravalent antibody were evaluated in an ELISA assay using an immobilized full length VEGF165-His protein (R & D Systems) and a human Ang-2-His protein (R & D Systems) for detection of bound bispecific antibody. Only the bispecific tetravalent < VEGF-Ang-2> antibody was able to bind VEGF and Ang-2 simultaneously and thus bridge both antigens, whereas the monospecific "standard" IgG1 antibody was unable to bind VEGF and Ang-2 simultaneously (fig. 7).

For this purpose, Falcon polystyrene clean-enhanced microtiter plates were coated with 100 μ l of 2 μ g/mL recombinant human VEGF165(R & D Systems) in PBS for 2 hours at room temperature or overnight at 4 ℃. The wells were washed three times with 300. mu.l PBST (0, 2% Tween 20) and blocked with 200. mu.l 2% BSA 0, 1% Tween20 for 30 minutes at room temperature, followed by three washes with 300. mu.l PBST. Serial dilutions of 100 μ L/well purified bispecific tetravalent antibody in pbs (sigma) were added to the wells and incubated on a microtiter plate shaker at room temperature for 1 hour. The wells were washed three times with 300. mu.l PBST (0, 2% Tween 20) and bound antibody was detected by adding 100. mu.l of 0.5. mu.g/ml human Ang-2-His (R & D Systems) in PBS. The wells were washed three times with 300. mu.l PBST (0, 2% Tween 20) and bound Ang-2 was detected with 100. mu.l of 0.5. mu.g/mL < Ang-2> mIgG 1-biotin antibody (BAM0981, R & D Systems) for 1 hour at room temperature. Unbound detection antibody was washed off three times with 300. mu.l PBST (0, 2% Tween 20) and bound antibody was detected at room temperature for 1 hour by adding 100. mu.l of 1: 2000 streptavidin-POD conjugate (Roche Diagnostics GmbH, Cat. No. 11089153) diluted 1: 4 in blocking buffer. Unbound streptavidin-POD conjugate was washed off 3-6 times with 300 μ L PBST (0, 2% Tween 20) and bound streptavidin-POD conjugate was detected by adding 100 μ L ABTS/well. The absorbance measurements were carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

Demonstration of simultaneous binding of bispecific tetravalent antibody < VEGF-Ang-2> to VEGF-A and Ang-2 by Biacore

To further corroborate the data from the bridging ELISA, additional assays were established to confirm simultaneous binding to VEGF and Ang-2 using surface plasmon resonance technology on a Biacore T100 instrument according to the following protocol and analyzed using the T100 software package (T100Control, version 2.01, T100Evaluation, version 2.01, T100Kinetics, version 1.01): ang-2 was captured at a capture level of 2000-1700 RUs in PBS, 0.005% (v/v) Tween20 running buffer via a five His antibody (five His-Ab without BSA, Qiagen No.34660) immobilized on a CM5 chip via amine coupling (without BSA). HBS-N buffer served as the running buffer during coupling, and activation was accomplished by a mixture of EDC/NHS. pentahis-Ab B SA-free capture antibody was diluted in coupling buffer NaAc, pH 4.5, c ═ 30 μ g/mL, and finally the still activated carboxyl groups were blocked by injection of 1M ethanolamine; ligand densities of 5000 and 17000 RU were tested. Diluted with running buffer +1mg/mL BSA at a flow of 5. mu.L/min, Ang-2 with a concentration of 500nM was captured by the pentaHis-Ab. Subsequently, the binding of < Ang-2, VEGF > bispecific tetravalent antibodies to Ang-2 and to VEGF was demonstrated by incubation with rhVEGF and formation of sandwich complexes. For this purpose, the bispecific tetravalent < VEGF-Ang-2> antibody binds Ang-2 at a flow of 50 μ L/min and a concentration of 100nM (diluted with running buffer +1mg/mL BSA), and simultaneous binding is detected by incubation with VEGF (rhVEGF, R & D-Systems catalog number 293-VE) in PBS + 0.005% (v/v) Tween20 running buffer at a flow of 50 μ L/min and a VEGF concentration of 150 nM. The association time was 120 seconds and the dissociation time 1200 seconds. Regeneration was accomplished after each cycle with 2x10mM glycine pH 2.0 at 50 μ L/min flow and 60 seconds contact time. The sensorgram was corrected using the usual dual reference (control reference: binding of bispecific antibody and rhVEGF to the capture molecule pentahisab). The blank for each antibody was measured with rhVEGF concentration "0".

Generation of HEK293-Tie2 cell line

To determine that < Ang-2, VEGF > bispecific tetravalent antibodies interfere with Ang-2 stimulated Tie2 phosphorylation and Ang-2 binding to Tie2 on cells, a recombinant HEK293-Tie cell line was generated. Briefly, pcDNA 3-based plasmid (RB22-pcDNA3Topo hTie2) encoding full-length human Tie2 under the control of the CMV promoter and neomycin resistance marker was transfected into HEK293 cells (ATCC) using Fugene (Roche Applied science) as transfection reagent and resistant cells were selected in DMEM 10% FCS, 500. mu.g/ml G418. Individual clones were isolated via cloning cylinders and subsequently analyzed for Tie2 expression by FACS. Clone 22 was identified as a clone with high and stable Tie2 expression even in the absence of G418 (HEK293-Tie2 clone 22). Subsequently, cell assays were performed using HEK293-Tie2 clone 22: ang-2 induced Tie2 phosphorylation and Ang-2 cell ligand binding assays.

Ang-2 induced Tie2 phosphorylation assay

Measured according to the following assay principle<Ang-2，VEGF>Inhibition of Ang-2 induced Tie2 phosphorylation by bispecific tetravalent antibodies. HEK293-Tie2 clone 22 was stimulated with Ang-2 for 5 minutes in the absence or presence of Ang-2 antibody and P-Tie2 was quantified by sandwich ELISA. Briefly, 2 × 10 per well⁵The individual HEK293-Tie2 clone 22 cells were cultured overnight in 100. mu.l DMEM, 10% FCS, 500. mu.g/ml geneticin poly-D-lysine coated 96-well microtiter plates. The following day, preparation in microtiter plates<Ang-2，VEGF>Titration of bispecific tetravalent antibody was performed (4-fold concentrated, 75. mu.l final volume/well in duplicate) and was matched with 75. mu.l Ang-2 (R)&D systems#623-AN]Dilutions (3.2. mu.g/ml as 4-fold concentrated solution) were mixed. The antibody and Ang-2 were preincubated for 15 minutes at room temperature. Mu.l of the mixture was added to HEK293-Tie2 clone 22 cells (with 1mM NaV)₃O₄Sigma # S6508 were preincubated together for 5 minutes) and incubated at 37 ℃ for 5 minutes. Subsequently, cells were plated with 200. mu.l of ice-cold PBS +1mM NaV per well₃O₄Washed and lysed by adding 120. mu.l lysis buffer (20mM Tris, pH 8.0, 137 mM) per well on iceNaCl, 1% NP-40, 10% glycerol, 2mM EDTA, 1mM NaV₃O₄1mM PMSF and 10. mu.g/ml Aprotinin (Aprotinin)). Cells were lysed at 4 ℃ for 30 min on a microtiter plate shaker, and 100. mu.l of lysate were transferred directly into p-Tie2 ELISA microtiter plates without prior centrifugation and without total protein determination (R-Tie 2)&DSystems，R&D # DY 990). The amount of P-Tie2 was quantified according to the manufacturer's instructions and the IC50 value of inhibition was determined using the XLfit4 analysis plug-in program (dose response one site, type 205) by Excel. IC50 values may be compared within one experiment, but there may be variations between experiments.

VEGF-induced HUVEC proliferation assay

VEGF-induced proliferation of HUVECs (human umbilical vein endothelial cells, Promocell # C-12200) was selected to measure<Ang-2，VEGF>Cell function of bispecific tetravalent antibody. Briefly, 5000 HUVEC cells per 96 wells (low passage number, 5 passages or less) were incubated in collagen I coated BD Biocoat collagen I96 well microtiter plates (BD #354407/35640) overnight in 100. mu.l starvation medium (EBM-2 endothelial basal medium 2, Promocell # C-22211, 0.5% FCS, penicillin/streptomycin). Will vary in concentration<Ang-2，VEGF>The bispecific tetravalent antibody was mixed with rhVEGF (30ngl/ml final concentration, BD #354107) and preincubated for 15 min at room temperature. Subsequently, the mixture was added to HUVEC cells and they were incubated at 37 ℃ with 5% CO₂Incubate together for 72 hours. On the day of analysis, the plates were equilibrated to room temperature for 30 minutes and cell viability/proliferation was determined using the CellTiter-GloTM luminescent cell viability assay kit according to the manual (Promega, # G7571/2/3). Luminescence was measured in a spectrometer.

Example 1

Generation, expression, purification and characterization of bispecific and tetravalent antibodies recognizing Ang-2 and VEGF-a

In a first example, a bispecific tetravalent antibody without a linker between the respective antibody chains recognizing Ang-2 and VEGF-a was generated by fusing a VH-CL domain fusion against VEGF-a via the (G4S) 4-linker to the C-terminus of the heavy chain of an antibody recognizing Ang-2 (SEQ1 or the corresponding IgG1 allotype). To obtain bispecific tetravalent antibodies, this heavy chain construct was co-expressed with a plasmid encoding the corresponding light chain of the Ang-2 antibody (SEQ3) and a VL-CH1 domain fusion recognizing VEGF-a (SEQ 2). The scheme for the corresponding antibody is given in figure 5.

Bispecific tetravalent antibodies were generated by classical molecular biology techniques as described in the general methods section and transiently expressed in HEK293F cells, as described above. Subsequently, it was purified from the supernatant by a combination of protein a affinity chromatography and size exclusion chromatography. The obtained products were characterized in terms of identity (by mass spectrometry) and in terms of analytical characteristics such as purity (by SDS-PAGE), monomer content and stability.

These data show that bispecific tetravalent antibodies can be produced in good yield and are stable.

Subsequently, the binding and simultaneous binding of Ang-2 and VEGF-a was investigated by ELISA and Biacore assays described above, and functional properties such as inhibition of Tie2 phosphorylation and inhibition of VEGF-induced HUVEC proliferation were analyzed, showing that the generated bispecific tetravalent antibody was able to bind Ang-2 and VEGF-a while blocking their activity.

Example 2

Generation, expression, purification and characterization of bispecific and tetravalent antibodies recognizing Ang-2 and VEGF-a

In a second example, a bispecific tetravalent antibody without a linker between the respective antibody chains recognizing Ang-2 and VEGF-a was generated by fusing a VH-CL domain fusion against VEGF-a via the (G4S) 4-linker to the N-terminus of the heavy chain of an antibody recognizing Ang-2 (SEQ4 or the corresponding IgG1 allotype). To obtain bispecific tetravalent antibodies, this heavy chain construct was co-expressed with a plasmid encoding the corresponding light chain of the Ang-2 antibody (SEQ3) and a VL-CH1 domain fusion recognizing VEGF-a (SEQ 2). The scheme for the corresponding antibody is given in figure 6.

Bispecific tetravalent antibodies were generated by classical molecular biology techniques as described in the general methods section and transiently expressed in HEK293F cells, as described above. Subsequently, it was purified from the supernatant by a combination of protein a affinity chromatography and size exclusion chromatography. The obtained products were characterized in terms of identity (by mass spectrometry) and in terms of analytical characteristics such as purity (by SDS-PAGE), monomer content and stability.

These data show that bispecific tetravalent antibodies can be produced in good yield and are stable.

Subsequently, the binding and simultaneous binding of Ang-2 and VEGF-a was investigated by ELISA and Biacore assays described above, and functional properties such as inhibition of Tie2 phosphorylation and inhibition of VEGF-induced HUVEC proliferation were analyzed, showing that the generated bispecific tetravalent antibody was able to bind Ang-2 and VEGF-a while blocking their activity.

Example 3

Generation, expression, purification and characterization of bispecific and tetravalent antibodies recognizing Ang-2 and VEGF-a

In a third example, a bispecific tetravalent antibody without a linker between the respective antibody chains recognizing Ang-2 and VEGF-a was generated by fusing the VH-CH1Fab domain for Ang-2 via the (G4S) 4-linker to the C-terminus of the heavy chain of a CH1-CL exchange antibody recognizing VEGF (SEQ5 or the corresponding IgG1 allotype). To obtain bispecific tetravalent antibodies, this heavy chain construct was co-expressed with a plasmid encoding the corresponding light chain of the Ang-2 antibody (SEQ3) and a VL-CH1 domain fusion recognizing VEGF-a (SEQ 2). The scheme for the corresponding antibody is given in figure 7.

Bispecific tetravalent antibodies were generated by classical molecular biology techniques as described in the general methods section and transiently expressed in HEK293F cells, as described above. Subsequently, it was purified from the supernatant by a combination of protein a affinity chromatography and size exclusion chromatography. The obtained products were characterized in terms of identity (by mass spectrometry) and in terms of analytical characteristics such as purity (by SDS-PAGE), monomer content and stability.

These data show that bispecific tetravalent antibodies can be produced in good yield and are stable.

Subsequently, the binding and simultaneous binding of Ang-2 and VEGF-a was investigated by ELISA and Biacore assays described above, and functional properties such as inhibition of Tie2 phosphorylation and inhibition of VEGF-induced HUVEC proliferation were analyzed, showing that the generated bispecific tetravalent antibody was able to bind Ang-2 and VEGF-a while blocking their activity.

Claims (15)

1. A bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker at the C or N terminus of the heavy chain of a);

wherein in the Fab fragment the following modifications are performed:

in the two Fab fragments of a), or in the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other.

2. A bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker at the C or N terminus of the heavy chain of a);

wherein in the Fab fragment the following modifications are performed:

in the two Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and

the constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

or

The constant domains CL and CH1 replace each other.

3. A bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker at the C or N terminus of the heavy chain of a);

wherein in the Fab fragment the following modifications are performed:

in the two Fab fragments of a),

the variable domains VL and VH are replaced with each other,

or

The constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and

the constant domains CL and CH1 replace each other.

4. A bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker at the C or N terminus of the heavy chain of a);

wherein in the Fab fragment the following modifications are performed:

in the two Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and is

In the two Fab fragments of b),

the constant domains CL and CH1 replace each other.

5. A bispecific antigen binding protein comprising:

a) two light chains and two heavy chains of an antibody that specifically binds a first antigen and comprises two Fab fragments;

b) two additional Fab fragments of an antibody that specifically binds to a second antigen, wherein the additional Fab fragments are fused via a peptide linker at the C or N terminus of the heavy chain of a);

wherein in the Fab fragment the following modifications are performed:

in the two Fab fragments of a),

the constant domains CL and CH1 replace each other,

and is

In the two Fab fragments of b),

the variable domains VL and VH are substituted for each other.

6. The bispecific antigen binding protein according to claim 1, characterized in that:

in the Fab fragment, the following modifications were performed:

in the two Fab fragments of a),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other.

7. The bispecific antigen binding protein according to claim 6, characterized in that:

in the Fab fragment, the following modifications were performed:

in the two Fab fragments of a),

the constant domains CL and CH1 replace each other.

8. The bispecific antigen binding protein according to claim 1, characterized in that:

in the Fab fragment, the following modifications were performed:

in the two Fab fragments of b),

the variable domains VL and VH are replaced with each other,

and/or

The constant domains CL and CH1 replace each other.

9. The bispecific antigen binding protein according to claim 8, characterized in that:

in the Fab fragment, the following modifications were performed:

in the two Fab fragments of b),

the constant domains CL and CH1 replace each other.

10. The bispecific antigen binding protein according to any one of claims 1 to 9, wherein the additional Fab fragment is fused via a peptide linker to the C-terminus of the heavy chain of a), or to the N-terminus of the heavy chain of a).

11. A process for preparing a bispecific antigen binding protein according to any one of claims 1 to 10, comprising the steps of:

a) transforming a host cell with a vector comprising a nucleic acid molecule encoding a bispecific antigen binding protein according to any one of claims 1 to 10,

b) culturing said host cell under conditions that allow synthesis of said antigen binding protein molecule; and are

c) Recovering the antigen binding protein molecule from the culture.

12. A host cell comprising a vector according to claim 11.

13. A pharmaceutical composition comprising a bispecific antigen binding protein according to any one of claims 1 to 10 and at least one pharmaceutically acceptable excipient.

14. Use of a bispecific antigen binding protein according to any one of claims 1 to 10 in the manufacture of a medicament for the treatment of cancer.

15. Use of a bispecific antigen binding protein according to any one of claims 1 to 10 for the manufacture of a medicament for the treatment of a patient in need of such treatment, characterized in that a therapeutically effective amount of a bispecific antigen binding protein according to any one of claims 1 to 10 is administered to said patient.