HK1145845B

HK1145845B - Bivalent, bispecific antibodies

Info

Publication number: HK1145845B
Application number: HK11100048.9A
Authority: HK
Inventors: 克里斯蒂安‧克莱因; 沃尔夫冈‧舍费尔
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2007-12-21
Filing date: 2008-12-16
Publication date: 2013-07-12

Description

Bivalent, bispecific antibodies

The present invention relates to novel bivalent, bispecific antibodies, their preparation and use.

Background

Engineered proteins, such as bi-or multispecific antibodies capable of binding more than 2 antigens, are known in the art. Such multispecific binding proteins may be produced using cell fusion, chemical conjugation, or recombinant DNA techniques.

A wide variety of recombinant bispecific antibody formats have recently been developed, such as tetravalent bispecific antibodies by fusion of, for example, an IgG antibody format and a single chain domain (see, e.g., Coloma, M.J., et al, Nature Biotech. (Nature Biotech.) 15(1997) 159-163; WO 2001077342; and Morrison, S.L., Nature Biotech. (Nature Biotech.) 25(2007) 1233-1234).

Furthermore, several other novel forms have been developed which are capable of binding more than 2 antigens, wherein the central structure of the antibody (IgA, IgD, IgE, IgG or IgM) no longer holds for example diabodies, triabodies or tetrabodies, minibodies, several single-chain forms (scFv, Bis-scFv) (Holliger, P., et al, Nature Biotech (Nature Biotechnology) 23(2005) 1126-.

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

Ideally, therefore, one should aim to develop bispecific antibodies with a general structure very similar to naturally occurring antibodies (e.g. IgA, IgD, IgE, IgG or IgM) with minimal deviation from human sequences.

In one approach, bispecific antibodies very similar to native antibodies were generated using the cell hybridoma (quadroma) technique (see Milstein, c. and a.c. cuello, Nature, 305(1983)537-40) which is based on somatic fusion of two different hybridoma cell lines expressing a murine monoclonal antibody with the desired specificity for the bispecific antibody. Because of the random pairing of the two different antibody heavy and light chains in the resulting hybrid-hybridoma (cell-hybridoma) cell lines, up to 10 different antibody types were generated, only one of which was the desired functional bispecific antibody. This implies the need for complex purification procedures due to the presence of mismatched byproducts and significantly reduced yields (see, e.g., Morrison, S.L., Nature Biotech 25(2007) 1233-1234). In general, the same mismatch by-product problem remains if recombinant expression techniques are used.

A method for circumventing the problem of mismatched byproducts, termed "bulge-entry-holes" (knobs-into-holes), aims to force pairing of two different antibody heavy chains by introducing mutations into the CH3 domain to modify the contact interface. On one chain, a large volume of amino acids is replaced by amino acids with short side chains to form "holes". Conversely, an amino acid with a large side chain was introduced into another CH3 domain to form a "bulge". By co-expressing the two heavy chains (and the two identical light chains, which must be adapted to both heavy chains), a high yield of the heterodimeric form ('bulge-hole') compared to the homodimeric form ('hole-hole' or 'bulge-bulge') was observed (Ridgway, j.b., Presta, LG, Carter, P and WO 1996027011). The percentage of heterodimers can be further increased by rebuilding the interaction surface of the two CH3 domains using phage display and introducing disulfide bonds to stabilize the heterodimers (Merchant A.M, et al, Nature Biotech 16(1998) 677-cok 681; Atwell, S., Ridgway, J.B., Wells, J.A., Carter, P., J.mol.biol. (journal of molecular biology) 270(1997) 26-35). New methods for the projection-entry-hole technique are described, for example, in EP 1870459a 1. Although this format appears to be very attractive, no data currently exists that describes progress toward the clinic. An important constraint of this strategy is that the light chains of the two parent antibodies must be identical to prevent mismatches and the formation of inactive molecules. Thus, this technique is not suitable for the easy development of recombinant, bivalent bispecific antibodies against both antigens from two antibodies against the first and second antigens, since the heavy chains and/or the same light chains of these antibodies have to be optimized.

Simon T. et al, EMBO Journal (EMBO J), 9(1990)1051-1056, are directed to domain mutants of monospecific antibodies.

Summary of The Invention

The present invention relates to a bivalent, bispecific antibody comprising:

a) a light chain and a heavy chain of an antibody that specifically binds a first antigen; and

b) a light chain and a heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are replaced with each other.

Another embodiment of the invention is a method for the preparation of a bivalent, bispecific antibody according to the invention, comprising the following steps:

a) the host cell is transformed with the following items,

-a vector comprising nucleic acid molecules encoding the light and heavy chains of an antibody specifically binding to a first antigen

-a vector comprising nucleic acid molecules encoding the light and heavy chains of an antibody specifically binding to a second antigen, wherein the constant domains CL and CH1 are replaced by each other;

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

c) recovering the antibody molecule from the culture.

Another embodiment of the invention is a host cell comprising

A vector comprising nucleic acid molecules encoding the light and heavy chains of an antibody specifically binding to a second antigen, wherein the constant domains CL and CH1 are replaced by each other.

Another embodiment of the invention is a composition, preferably a pharmaceutical or diagnostic composition, of an antibody according to the invention.

Another embodiment of the invention is a pharmaceutical composition comprising an antibody according to the invention and at least one pharmaceutically acceptable excipient.

Another embodiment of the invention is a method for treating a patient in need of treatment, characterized in that a therapeutically effective amount of an antibody according to the invention is administered to said patient.

Detailed Description

The present invention relates to a bivalent, bispecific antibody comprising:

Thus, the bivalent, bispecific antibody comprises:

a) a first light chain and a first heavy chain of an antibody that specifically binds a first antigen; and

b) a second light chain and a second heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 of the second light chain and the second heavy chain are replaced with each other.

Thus, for the antibody that specifically binds to the second antigen, the following applies:

in the light chain

The constant light chain domain CL is replaced by the constant heavy chain domain CH1 of the antibody;

and in the heavy chain

The constant light chain domain CH1 was replaced by the constant light chain domain CL of the antibody.

The term "antibody" as used herein refers to an intact, monoclonal antibody. The intact antibody consists of two pairs of "light chains" (LC) and "heavy chains" (HC) (the Light Chain (LC)/heavy chain pair is abbreviated herein as LC/HC). The light and heavy chains of the antibody are polypeptides consisting of several domains. In a complete antibody, each heavy chain comprises a heavy chain variable region (abbreviated HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes heavy chain constant domains CH1, CH2, and CH3 (antibody types IgA, IgD, and IgG) and, optionally, heavy chain constant domain CH4 (antibody types IgE and IgM). Each light chain comprises a light chain variable domain VL and a light chain constant domain CL. The structure of one naturally occurring intact antibody, the IgG antibody, is shown, for example, in figure 1. The variable domains VH and VL can be further subdivided into hypervariable regions, termed Complementarity Determining Regions (CDRs), between which more conserved regions, termed Framework Regions (FRs), are distributed. Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4((Janeway, c.a., jr. et al., (2001) Immunobiology (immunology), 5 th edition, Garland press (Garland Publishing) and Woof, j., Burton D Nat rev immunol (review by nature) 4(2004)89-99) two pairs of heavy and light chains (HC/LC) are capable of specifically binding to the same antigen.

There are 5 mammalian antibody heavy chain types represented by greek letters: α, δ, ε, γ, and μ (Janeway, C.A., Jr., et al., (2001) Immunobiology, 5 th edition, Garland Press (Garland Publishing)). The type of heavy chain present defines the type of antibody; these chains are present in IgA, IgD, IgE, IgG, and IgM antibodies, respectively (Rhoades RA, Pflanzer RG (2002); Human Physiology, 4 th edition, thomson knowledge). The different heavy chains differ in size and composition; alpha and gamma contain about 450 amino acids, while mu and epsilon have about 550 amino acids.

Each heavy chain has two regions, a constant region and a variable region. The constant region is the same in all antibodies of the same isotype, but differs in antibodies of different isotypes. Heavy chains γ, α and δ have a constant region consisting of 3 constant domains CH1, CH2 and CH3 (in a line) and a hinge region for increased flexibility (Woof, j., Burton D Nat Rev Immunol (natural immunological review) 4(2004) 89-99); heavy chains μ and ∈ have constant regions consisting of 4 constant domains CH1, CH2, CH3 and CH4 (Janeway, c.a., jr., et al., (2001) immunology, 5 th edition, Garland Publishing company (Garland Publishing)). The variable region of the heavy chain differs among antibodies produced by different B cells, but is the same for all antibodies produced by a single B cell or B cell clone. The variable region of each heavy chain is about 110 amino acids long and consists of a single antibody domain.

In mammals, there are only two types of light chains, which are called λ and κ. The light chain has two contiguous domains: 1 constant domain CL and 1 variable domain VL. The approximate length of the light chain is 211-217 amino acids. Preferably, the light chain is a kappa light chain and the constant domain CL is preferably ck.

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

The "antibody" according to the invention may be of any type (e.g. IgA, IgD, IgE, IgG, and IgM, preferably IgG or IgE), or subtype (e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2, preferably IgG1), wherein the two antibodies from which the bivalent bispecific antibody according to the invention is derived have an Fc part of the same subtype (e.g. IgG1, IgG4, etc., preferably IgG1), preferably of the same allotype (e.g. caucasian).

The "Fc portion of an antibody" is a term well known to the skilled artisan and is defined based on the papain cleavage of the antibody. The antibodies according to the invention comprise, for example, an Fc part, preferably derived from human origin, and preferably all other parts of a human constant region. The Fc portion of the antibody is directly involved in complement activation, C1q binding, C3 activation and Fc receptor binding. Although the effect of antibodies on the complement system depends on certain conditions, binding to C1q results from a defined binding site in the Fc portion. Such binding sites are known in the art and described, for example, in Lukas, t.j., et al, j.immunol. (journal of immunology) 127(1981) 2555-2560; brunhouse, r., and Cebra, j.j., mol.immunol. (molecular immunology) 16(1979) 907-; burton, D.R., et al, Nature 288(1980) 338-344; thommesen, J.E., et al, mol.Immunol. (molecular immunology) 37(2000) 995-1004; idusogie, e.e., et al, j.immunol. (J.Immunol.) 164(2000) 4178-4184; hezareh, M., et al, J.Virol, (J.Virol) 75(2001) 12161-12168; morgan, A., et al, Immunology 86(1995) 319-324; and EP 0307434. Such binding sites are for example L234, L235, D270, N297, E318, K320, K322, P331 and P329 (EU catalogue numbering according to Kabat, see below). Antibodies of the subtypes IgG1, IgG2 and IgG3 generally show complement activation, C1q binding and C3 activation, whereas IgG4 does not activate the complement system, does not bind C1q and does not activate C3. Preferably, the Fc portion is a human Fc portion.

The term "chimeric antibody" refers to an antibody that includes a variable, i.e., binding, region from one source or species, and at least a portion of a constant region from a different source or species, typically prepared by recombinant DNA techniques. Chimeric antibodies comprising murine variable regions and human constant regions are preferred. Other preferred forms of "chimeric antibodies" encompassed by the present invention are those in which the constant regions have been modified or altered from the constant regions of the original antibody to produce properties according to the present invention, particularly with respect to C1q binding and/or Fc receptor (FcR) binding. Such "chimeric" antibodies are also referred to as "class switch antibodies". Chimeric antibodies are the product of an expressed immunoglobulin gene (including DNA segments encoding immunoglobulin variable regions and DNA segments encoding immunoglobulin constant regions). Methods for making chimeric antibodies include conventional recombinant DNA and gene transfection techniques that are currently well known in the art. See, for example, Morrison, S.L., et al, Proc. Natl. Acad. Sci. USA 81(1984) 6851-6855; US 5,202,238 and 5,204, 244.

The term "humanized antibody" refers to antibodies in which the framework or "complementarity determining regions" (CDRs) have been modified to include CDRs from an immunoglobulin that are specifically different from the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted onto 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 indicated above for the chimeric antibodies. Other forms of "humanized antibodies" encompassed by the present invention are those humanized antibodies in which the constant regions have additionally been modified or altered from the constant regions of the original antibody to produce properties according to the present 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., current chemical biology views (curr. opin. in chemical biology)5(2001) 368-. Human antibodies can also be produced in transgenic animals (e.g., mice) that, when immunized, are capable of producing all or part of a selected human antibody in the absence of endogenous immunoglobulin production. Transfer of a human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al, Proc. Natl. Acad. Sci. USA, Proc. Natl. Acad. Sci. 90 (1993)) 2551-. Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G., J.mol.biol. (J.Mol.Biol.) (J.M.biol.) 227(1992) 381-. The techniques of Cole et al and Boerner et al can also be used to prepare human Monoclonal antibodies (Cole et al, Monoclonal antibodies and Cancer Therapy, Alan R.Liss, p.77-96(1985), and Boerner, P.et al, J.Immunol.147 (1991) 86-95). As already mentioned for the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also includes antibodies which are modified in the constant region to produce the properties according to the invention, in particular with regard to C1q binding and/or Fc receptor (FcR) binding, for example by "class switching", i.e.by altering or mutating 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, produced or isolated by recombinant methods, 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 variable and constant regions in rearranged form. Recombinant human antibodies according to the invention have undergone somatic hypermutation in vivo. Thus, the amino acid sequences of the VH and VL regions of a recombinant antibody are sequences that, although derived from and related to human germline VH and VL sequences, may not naturally occur in vivo in human antibody germline repertoires.

"variable domain" (variable domain of a light chain (VL), variable region of a heavy chain (VH)) as used herein, denotes each pair of light and heavy chains that is directly involved in binding of an antibody to an antigen. The domains of variable human light and heavy chains have the same general structure and each domain comprises 4 Framework (FR) regions, the sequences of which are generally conserved, connected by 3 "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation and the CDRs may form loops connecting the β -sheet structures. The CDRs in each chain are held in their three-dimensional structure by 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 another object of the invention.

As used herein, the term "hypervariable region" or "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions comprise amino acid residues from the "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those variable domain regions other than the hypervariable region residues defined herein. Thus, the light and heavy chains of an antibody comprise, from N-terminus to C-terminus, the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR 4. The CDRs on each chain are separated by the framework amino acids. In particular, CDR3 of the heavy chain is the region most contributing to antigen binding. CDR and FR regions are defined according to the standard definition of the sequence of an immunologically significant protein sequence (Sequences of Proteins of immunological Interest), 5 th edition, public Health services, National Institutes of Health, Bethesda, Md. (1991), by Kabat et al.

The "constant domains" of the heavy and light chains are not directly involved in binding the antibody to the antigen but exhibit a variety of effector functions. Antibodies or immunoglobulins are classified into the following classes according to the amino acid sequence of their heavy chain constant region:

the term "bivalent bispecific antibody" as used herein refers to an antibody as described above, wherein each of the two pairs of heavy and light chains (HC/LC) specifically binds to a different antigen, i.e. the first heavy chain and the first light chain (derived from an antibody against a first antigen) specifically bind together to a first antigen, and the second heavy chain and the second light chain (derived from an antibody against a second antigen) specifically bind together to a second antigen (as shown in figure 2); the bivalent, bispecific antibody is capable of specifically binding to two different antigens simultaneously, and not more than two antigens simultaneously, in contrast to monospecific antibodies which are capable of binding to only one antigen on the one hand and tetravalent, tetraspecific antibodies which are capable of binding to four antigen molecules simultaneously, for example, on the other hand.

According to the present invention, the ratio of the desired bivalent bispecific antibody to the undesired side products can be increased by replacing certain domains in only one pair of heavy and light chains (HC/LC). A first of the two pairs of HC/LC pairs is derived from an antibody that specifically binds a first antigen and remains substantially unchanged, while a second of the two pairs of HC/LC pairs is derived from an antibody that specifically binds a second antigen and is altered by the following substitutions:

-light chain:replacing the constant light chain domain CL with the constant heavy chain domain CH1 of the antibody that specifically binds a second antigen, and

-heavy chain:the constant heavy chain domain CH1 was replaced with the constant light chain domain CL of the antibody that specifically binds the second antigen.

Thus the bivalent bispecific antibody thus generated is an artificial antibody comprising

b) a light chain and a heavy chain of an antibody that specifically binds a second antigen;

wherein the light chain (of an antibody that specifically binds a second antigen) comprises the constant domain CH1 instead of CL,

and is

Wherein the heavy chain (of an antibody that specifically binds a second antigen) comprises the constant domain CL instead of CH 1.

In another aspect of the invention, such an increased ratio of the desired bivalent bispecific antibody to undesired side products can be further increased by one of the following two alternatives:

A) first alternative (see fig. 3):

the CH3 domain of the bivalent bispecific antibody according to the invention may be altered by the "bulge-entry-hole" technique, as described, for example, in WO 96/027011, Ridgway J.B., et al, Protein Eng 9 (Protein engineering) 1996) 617-621; and Merchant, A.M., et al, NatBiotechnol (NatBiotechnol) 16(1998) 677-681. In this method, the interaction surface of the two CH3 domains is altered to increase heterodimerization of the two heavy chains comprising the two CH3 domains. One of the two CH3 domains (of both heavy chains) may be "bulge" and the other "hole". The introduction of disulfide bonds stabilizes the heterodimer (Merchant, A.M, et al, Nature Biotech 16(1998) 677-.

Thus in a preferred embodiment, the CH3 domain of the bivalent, bispecific antibody is altered by a "bulge-in-hole" technique, in which the first CH3 domain and the second CH3 domain are each contacted at an interface comprising the initial interface between antibody CH3 domains, the "bulge-entry-hole" technique involves further stabilization by the introduction of disulfide bonds into the CH3 domain (described in WO 96/027011, Ridgway, J.B., et al, Protein Eng (Protein engineering) 9(1996) 617-621; Merchant, A.M., et al, Nature Biotech (Nature Biotech) 16(1998) 677-681; and Atwell, S., Ridgway, J.B., Wells, J.A., Carter, P., J.mol.biol. (journal of molecular biology) 270(1997)26-35) to facilitate the formation of bivalent bispecific antibodies.

Thus, in one aspect of the invention, the bivalent, bispecific antibody is characterized

The CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are each contacted at an interface that includes the initial interface between the antibody CH3 domains;

wherein the interface is altered to facilitate formation of a bivalent bispecific antibody, wherein the alteration is characterized by:

a) the CH3 domain of one heavy chain is altered,

thus, within the initial interface of the CH3 domain of one heavy chain in contact with the initial interface of the CH3 domain of the other heavy chain in a bivalent bispecific antibody,

the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a bulge within the interface of the CH3 domain of one heavy chain, which bulge may be positioned in a cavity within the interface of the CH3 domain of the other heavy chain

And is

b) The CH3 domain of the other heavy chain is altered,

thus, within the initial interface of the second CH3 domain in contact with the initial interface of the first CH3 domain in a bivalent bispecific antibody,

the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the interface of the second CH3 domain into which a bulge within the interface of the first CH3 domain can be located.

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

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

In one aspect of the invention, the two CH3 domains were further altered to introduce cysteine (C) as the amino acid at the corresponding position of each CH3 domain, thereby allowing disulfide bond formation between the two CH3 domains.

In another preferred embodiment of the invention, the residue is obtained by using residue R409D for the bulge residue; K370E (K409D) and D399K for the hole residue; E357K to alter the two CH3 domains as described in, for example, EP 1870459a 1.

Or

B) Second alternative (see fig. 4):

by replacing one constant heavy chain domain CH3 with constant heavy chain domain CH 1; and the other constant heavy chain domain CH3 is replaced with a constant light chain domain CL. The constant heavy chain domain CH1 replacing the heavy chain domain CH3 may be of any Ig class (e.g., IgA, IgD, IgE, IgG, and IgM), or subtype (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2). The constant light chain domain CL replacing the heavy chain domain CH3 may be of the lambda or kappa type, preferably of the kappa type.

Accordingly, a preferred embodiment of the invention is a bivalent, bispecific antibody comprising:

b) a light chain and a heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are replaced with each other,

and wherein, optionally, the first and second substrates are,

c) the CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are each contacted at an interface that includes the initial interface between the antibody CH3 domains;

ca) changes the CH3 domain of one heavy chain,

And is

cb) alters the CH3 domain of the other heavy chain,

the amino acid residue is replaced with an amino acid residue having a smaller side chain volume, thereby creating a cavity within the interface of the second CH3 domain into which a bulge within the interface of the first CH3 domain can be located;

or d)

One constant heavy chain domain CH3 was replaced by constant heavy chain domain CH 1; and the other constant heavy chain domain CH3 is replaced by a constant light chain domain CL.

The terms "antigen" or "antigenic molecule" as used herein, are used interchangeably and refer to all molecules capable of being specifically bound by an antibody. A bivalent bispecific antibody specifically binds a first antigen and a second, different antigen. The term "antigen" as used herein includes, for example, proteins, different epitopes on proteins (as different antigens within the meaning of the invention) and polysaccharides. This includes mainly parts of bacteria, viruses and other microorganisms (coat, envelope, cell wall, flagella, pili and toxins). Lipids and nucleic acids are antigenic only when bound to proteins and polysaccharides. Non-microbial foreign (non-self) antigens may include pollen, egg white, and proteins on the surface of transplanted or infused blood cells from transplanted tissues and organs. Preferably, the antigen is selected from the group consisting of cytokines, cell surface proteins, enzymes and receptors.

Tumor antigens are those antigens which are presented by MHC I or MHC II molecules on the surface of tumor cells. These antigens can sometimes be presented by tumor cells and never by normal cells. As such, they are referred to as Tumor Specific Antigens (TSAs) and are typically generated by tumor specific mutations. More common are antigens presented by tumor cells and normal cells, and they are called Tumor Associated Antigens (TAAs). Cytotoxic T lymphocytes recognizing these antigens may be able to destroy tumor cells before they proliferate or metastasize. Tumor antigens may also be present on the tumor surface in the form of, for example, mutated receptors, in which case they should be recognized by B cells.

In a preferred embodiment, at least one of the two different antigens (first and second antigen) to which the bivalent, bispecific antibody specifically binds is a tumor antigen.

In another preferred embodiment, the two different antigens (the first and second antigens) to which the bivalent, bispecific antibody specifically binds are both tumor antigens; in this case, the first and second antigens may also be two different epitopes on the same tumor-specific protein.

In another preferred embodiment, one of the two different antigens (first and second antigen) to which the bivalent, bispecific antibody specifically binds is a tumor antigen and the other is an effector cell antigen, e.g. T-cell receptor, CD3, CD16 etc.

In another preferred embodiment, one of the two different antigens (first and second antigen) to which the bivalent, bispecific antibody specifically binds is a tumor antigen and the other is an anti-cancer agent such as a toxin or a kinase inhibitor.

As used herein, "specifically binds" or "specifically binds to … …" refers to an antibody that specifically binds an antigen. Preferably, the binding affinity of an antibody that specifically binds to the antigen is a KD-value of 10^-9mol/l or less (e.g., 10)^-10mol/l), preferably has a KD-value of 10^-10mol/l or less (e.g., 10)^-12mol/l). Binding affinity Using standard binding assays, such as surface plasmon resonance (Biacore) technique) To be determined.

The term "epitope" includes any polypeptide determinant capable of specifically binding to an antibody. In certain embodiments, epitope determinants include chemically active surface components (groupings) of molecules, such as amino acids, sugar side chains, phosphoryl groups, or sulfonyl groups, which, in certain embodiments, may have specific three-dimensional structural characteristics, and/or specific charge characteristics. An epitope is a region of an antigen that is bound by an antibody. In certain embodiments, an antibody is said to specifically bind to an antigen when it preferentially recognizes its target antigen in a complex mixture of proteins and/or macromolecules.

Another embodiment of the invention is a method for the preparation of a bivalent, bispecific antibody according to the invention, comprising

a) The host cell is transformed with the following items,

c) recovering the antibody molecule from the culture.

Typically, there are two vectors encoding the light and heavy chains of the antibody that specifically binds to the first antigen, and two additional vectors encoding the light and heavy chains of the antibody that specifically binds to the second antigen. One of the two vectors encodes a respective light chain and the other of the two vectors encodes a respective heavy chain. However, in another method for preparing a bivalent bispecific antibody according to the present invention, a host cell may be transformed with only one first vector encoding the light and heavy chains of an antibody specifically binding to a first antigen and only one second vector encoding the light and heavy chains of an antibody specifically binding to a second antigen.

The invention includes methods for producing the antibodies, comprising culturing the respective host cells under conditions permitting synthesis of the antibody molecules and recovering the antibodies from the culture, e.g., by expressing

-a first nucleic acid sequence encoding a light chain of an antibody that specifically binds a first antigen;

-a second nucleic acid sequence encoding the heavy chain of the antibody that specifically binds to the first antigen;

-a third nucleic acid sequence encoding a light chain of an antibody that specifically binds to a second antigen, wherein the constant light chain domain CL is replaced by the constant heavy chain domain CH 1; and

-a fourth nucleic acid sequence encoding the heavy chain of the antibody that specifically binds to the second antigen, wherein the constant heavy chain domain CH1 is replaced by a constant light chain domain CL.

Another embodiment of the invention is a host cell comprising

a) Vectors comprising nucleic acid molecules encoding the light chain of an antibody that specifically binds to a first antigen and vectors comprising nucleic acid molecules encoding the heavy chain of an antibody that specifically binds to a first antigen

b) A vector comprising a nucleic acid molecule encoding a light chain of an antibody that specifically binds to a second antigen and a vector comprising a nucleic acid molecule encoding a heavy chain of an antibody that specifically binds to a second antigen, wherein constant domains CL and CH1 are replaced with each other.

Another embodiment of the invention is a composition, preferably a pharmaceutical or diagnostic composition, of a bivalent, bispecific antibody according to the invention.

Another embodiment of the invention is a pharmaceutical composition comprising a bivalent, bispecific antibody according to the invention and at least one pharmaceutically acceptable excipient.

Another embodiment of the present invention is a method for the treatment of a patient in need of such treatment, characterized in that a therapeutically effective amount of a bivalent bispecific antibody according to the present invention is administered to said patient.

The term "nucleic acid or nucleic acid molecule" as used herein is intended to include both DNA molecules and RNA molecules. The nucleic acid molecule may be single-stranded or double-stranded, but is preferably double-stranded DNA.

As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all of these designations include progeny. Thus, the words "transformant" and "transformed cell" include the primary subject cell and the culture from which it was derived, regardless of the number of transfers. It is also understood that the DNA content of all progeny may not be exactly consistent due to deliberate or inadvertent mutations. Variant progeny selected for the same function or biological activity in the originally transformed cell are included. Where different names are intended, they will be clear from the context.

The term "transformation" as used herein refers to the process of transferring a vector/nucleic acid into a host cell. If cells without a difficult cell wall barrier are used as host cells, transfection is carried out, for example, by the calcium phosphate precipitation method as described by Graham and van der Eh, Virology (Virology) 52(1978)546 ff. However, other methods of introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing a substantial cell wall structure are used, one method of transfection is, for example, calcium treatment with calcium chloride, as described by Cohen, F.N, et al, PNAS.69(1972)7110 ff.

The recombinant production of antibodies by transformation is well known in the art and is described, for example, in review articles Makrides, S.C., Protein Expr. Purif, (Protein Experimental purification) 17(1999) 183-202; geisse, s., et al, Protein expr. purif. (Protein experimental purification) 8(1996) 271-282; kaufman, R.J., mol.Biotechnol. (molecular Biotechnology) 16(2000) 151-161; werner, R.G., et al, Arzneimitelforschung 48(1998)870-880 and U.S. Pat. Nos. 6,331,415 and 4,816,567.

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 referred to as a 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 the mRNA.

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

An "expression vector" is a polynucleotide that, upon introduction into a suitable host cell, is capable of being transcribed and translated into a polypeptide. An "expression system" generally refers to a suitable host cell that includes an expression vector that functions to produce a desired expression product.

The bivalent bispecific antibody according to the invention is preferably generated by recombinant means. Such methods are generally known in the art and include protein expression in prokaryotic and eukaryotic cells followed by isolation of the antibody polypeptide and usually purification to pharmaceutical purity. For protein expression, nucleic acids encoding the light and heavy chains, or fragments thereof, are inserted into the 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, yeast or E.coli (E.coli) cells, and the antibody is recovered from the cells (supernatant or cells after lysis). The bivalent bispecific antibody may be present as intact cells, as a cell lysate or in partially purified or substantially pure form. Purification is carried out by standard techniques, including alkali/SDS treatment, column chromatography and other techniques known in the art, to eliminate other cellular components or other contaminants, such as other cellular nucleic acids or proteins. See Ausubel, f., et al, ed., Current protocol Molecular Biology (Current Molecular Biology protocol), Greene Publishing and Wiley interscience (Greene published and Wiley cross science), new york (1987).

Expression in NS0 cells is described, for example, in Barnes, L.M., et al, Cytotechnology 32(2000) 109-123; and Barnes, L.M., et al, Biotech.Bioeng. (Biotechnology and bioengineering) 73(2001) 261-. Transient expression is described, for example, in Durocher, y., et al, nucleic acids. res. (nucleic acids research) 30(2002) E9. Cloning of variable domains is described in Orlandi, R, et al, Proc.Natl.Acad.Sci.USA (proceedings of the national academy of sciences USA) 86(1989) 3833-3837; carter, p., et al, proc.natl.acad.sci.usa 89(1992) 4285-; and Norderhaug, l., et al, j.immunol. methods (journal of immunological methods) 204(1997) 77-87. Preferred transient expression systems (HEK 293) are described in Schlaeger, E.J., and Christensen, K.K., Cytology 30(1999)71-83 and Schlaeger, E.J., J.Immunol. methods 194(1996) 191-199.

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

A nucleic acid is "operably linked" when placed into 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, provided that 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, provided that it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence, provided that it is positioned to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading frame. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If the site is not present, the synthetic oligonucleotide adapter or linker is used according to conventional practice.

The bivalent bispecific antibody is suitably isolated from the culture medium by conventional immunoglobulin purification procedures such as, for example, protein a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. DNA and RNA encoding the monoclonal antibodies are readily isolated and sequenced using conventional procedures. Hybridoma cells can function as the source of DNA and RNA. Once isolated, the DNA may be inserted into an expression vector that is subsequently transfected into a host cell that does not otherwise produce immunoglobulins, such as a HEK293 cell, CHO cell, or myeloma cell, to obtain synthesis of recombinant monoclonal antibodies in the host cell.

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

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 understood that variations may be made to the procedures described without departing from the spirit of the invention.

Sequence listing

SEQ ID NO: 1 amino acid sequence of the heavy chain of a wild-type < IGF-1R > antibody

SEQ ID NO: 2 amino acid sequence of wild type < IGF-1R > antibody light chain

SEQ ID NO：3<IGF-1R>Heavy chain of CL-CH1 exchange antibody^*(HC^*) Amino acid sequence ofColumn, in which the heavy chain domain CH1 is replaced with the light chain domain CL.

SEQ ID NO：4<IGF-1R>Light chain of CL-CH1 exchange antibody^*(LC^*) Wherein the light chain domain CL is replaced with the heavy chain domain CH 1.

SEQ ID NO: amino acid sequence of 5 IGF-1R extracellular domain His-streptavidin binding peptide-tag (IGF-1R-His-SBP ECD)

SEQ ID NO: 6 amino acid sequence of wild-type ANGPT2< ANGPT2> antibody heavy chain

SEQ ID NO: 7 amino acid sequence of wild-type ANGPT2< ANGPT2> antibody light chain

SEQ ID NO: 8 amino acid sequence of CH3 Domain with T366W exchange (bulge) for use in bulge-entry-hole technology

SEQ ID NO: 9 amino acid sequence with CH3 Domain (well) exchanged T366S, L368A, Y407V for use in bulge-entry-well technology

SEQ ID NO: amino acid sequence of 10 IGF-1R extracellular domain His-streptavidin binding peptide-tag (IGF-1R-His-SBP ECD)

Drawings

FIG. 1 is a schematic representation of an IgG, a naturally occurring whole antibody specific for an antigen having two pairs of heavy and light chains comprising variable and constant domains in typical order.

Figure 2 schematic representation of a bivalent bispecific antibody comprising: a) a light chain and a heavy chain of an antibody that specifically binds a first antigen; and b) a light chain and a heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are replaced with each other.

Figure 3 schematic representation of a bivalent bispecific antibody comprising: a) a light chain and a heavy chain of an antibody that specifically binds a first antigen; and b) a light chain and a heavy chain of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are replaced by each other, and wherein the CH3 domains of both heavy chains are altered by a bulge-entry-hole technique.

Figure 4 schematic representation of a bivalent bispecific antibody comprising: a) a light chain and a heavy chain of an antibody that specifically binds a first antigen; and b) light and heavy chains of an antibody that specifically binds a second antigen, wherein the constant domains CL and CH1 are replaced by each other, and wherein one of the constant heavy chain domains CH3 of both heavy chains is replaced by the constant heavy chain domain CH1 and the other constant heavy chain domain CH3 is replaced by the constant light chain domain CL.

FIG. 5<IGF-1R>Heavy chain of CL-CH1 exchange antibody (with kappa constant light chain Domain CL)^**<IGF-1R>HC^**Protein sequence diagram of

FIG. 6<IGF-1R>Light chain of CL-CH1 exchange antibody^**<IGF-1R>LC^**Protein sequence diagram of

FIG. 7 heavy chain^**<IGF-1R>HC^**Expression vector pUC-HC^*Plasmid map of IGF-1R

FIG. 8 light chain^**<IGF-1R>LC^**Expression vector pUC-LC^*Plasmid map of IGF-1R

FIG. 94700-Hyg-OriP expression vector plasmid map

FIG. 10 assay principle of cellular FACSIMGF-1R-ANGPT 2 bridging assay for I24 IGF-1R expressing cells for detecting the presence of functional bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody

FIG. 11 demonstrates IGF-1R ECD Biacore

FIG. 12 purified with HC^*And LC^*Monospecific bivalent of (2)<IGF-1R>CL-CH1 exchange antibody (IgG 1)^*) SDS-PA of (2)GE and size exclusion chromatography, which was isolated from cell culture supernatants after transient transfection of HEK293-F cells.

FIG. 13 binding of monospecific < IGF-1R > CL-CH1 exchange antibody and wild type < IGF-1R > antibody to IGF-1R ECD in an ELISA based binding assay.

FIG. 14 SDS-PAGE and size exclusion chromatography of a mixture of < ANGPT2-IGF-1R > CL-CH1 exchange antibodies purified from cell culture supernatants from transiently transfected HEK293-F cells.

FIG. 15 results of samples A-F of a cellular FACSGF-1R-ANGPT 2 bridging assay performed on I24 IGF-1R expressing cells to detect the presence of functional bispecific < ANGPT2-IGF-1R > CL-CH1 exchanged antibody in the purified antibody mixture: purified protein samples a-F:

a is untreated I24

B ═ I24+2 μ g/mL hANGPT2+ hIgG isotype

C-I24 +2 μ g/mL hANGPT2+ from < IGF-1R > CL-CH1 exchange

Co-expression of antibodies and < ANGPT2> wild-type antibodies, including bispecific

< mixture of ANGPT2-IGF-1R > CL-CH1 exchange antibodies

D: is absent from

I24+2 μ g/mL wild-type antibody hANGPT2+ < ANGPT2 ≧ E ═ I24+2 μ g/mL

F-I24 + 2. mu.g/mL hANGPT2+ < IGF-1R > wild-type antibody

Examples

Materials and general methods

General information on the nucleotide Sequences of human immunoglobulin light and heavy chains is provided in Kabat, e.a., et al, Sequences of Proteins of immunological interest (Sequences of Proteins of immunological interest), 5 th edition, Public Health services, National Health institute (Public Health Service, National Institutes of Health), Bethesda, MD. (1991). Amino acids of an antibody chain are numbered and referenced according to EU numbering (Edelman, g.m., et al, proc.nat. acad.sci.usa 63(1969) 78-85; Kabat, e.a., et al, immunologically significant protein Sequences (Sequences of Proteins of Immunological Interest), 5 th edition, Public Health services, National Health institute (Public Health Service, National institute of Health), Bethesda, MD. (1991)).

Recombinant DNA technology

DNA is manipulated using standard methods, such as Sambrook, j, et al, Molecular cloning: (iii) Alabotory manual (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 segments are prepared from oligonucleotides prepared by chemical synthesis. The 600-1800bp long gene segment flanking the single restriction endonuclease cleavage site was assembled by annealing and ligation of oligonucleotides, including PCR amplification, and subsequently cloned into pPCRScript (Stratagene) of a pGA 4-based cloning vector through the indicated restriction sites, e.g., KpnI/SacI or AscI/PacI. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. The gene synthesis fragments were ordered according to the given instructions of Geneart (Regensburg, Germany).

DNA sequencing

The DNA sequence was determined by double-strand sequencing performed in MediGenomix GmbH (Martinsried, Germany) or Sequiserve GmbH (Vaterstetten, Germany).

DNA and protein sequence analysis and sequence data management

The software package version 10.2 of GCG (Genetics Computer Group, Madison, Wis.) and the Infmax Vector NT1 advanced Group version 8.0 are used for sequence construction, mapping, analysis, annotation and description.

Expression vector

For expression of the antibodies, variants of expression plasmids for transient expression in cells based on cDNA organization with the CMV-intron A promoter or on genomic organization with the CMV promoter (e.g.in HEK293EBNA or HEK293-F) are used.

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

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

-a beta-lactamase gene conferring ampicillin resistance in E.coli. The transcription unit of the antibody gene consists of the following elements:

unique restriction sites at the 5' end

Immediate early enhancer and promoter from human cytomegalovirus,

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

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

an immunoglobulin heavy chain signal sequence,

human antibody chains (wild type or with domain exchanges) as cDNA or as genomic organization with immunoglobulin exon-intron organization

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

-a unique restriction site at the 3' end.

The fusion genes comprising the antibody chains as described below are produced by PCR and/or gene synthesis and assembled using known recombinant methods and techniques by ligating the corresponding nucleic acid segments in various vectors, for example, using unique restriction sites. The subcloned nucleic acid sequences were verified 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 as described in Current Protocols in Cell Biology (Current Protocols for Cell Biology) (2000), Bonifacino, J.S., Dasso, M., Harford, J.B., Lippincott-Schwartz, J. and Yamada, K.M (eds.), John Wiley & Sons, Inc.

Bispecific antibodies were expressed by transient co-transfection of various expression plasmids in adherently grown HEK293-EBNA or in suspension grown HEK29-F cells, as described below.

Transient transfection in the HEK293-EBNA System

Bispecific antibodies were expressed by transient co-transfection of various expression plasmids (e.g., encoding heavy and modified heavy chains, and the corresponding light and modified light chains) in adherently grown HEK293-EBNA cells (EB virus nuclear antigen expressing human embryonic kidney cell line 293; American type culture center, accession No. ATCC # CRL-10852, Lot.959218) 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)). For transfection, FuGENE^TM6 transfection reagent (Roche Molecular Biochemicals)) according to FuGENE^TMThe ratio of the reagent (. mu.l) to DNA (. mu.g) was 4: 1 (in the range of 3: 1 to 6: 1). Proteins were expressed separately from each plasmid using plasmids encoding (modified and wild-type) light and heavy chains at a molar ratio of 1: 1 (equimolar) in the range of 1: 2 to 2: 1. On day 3, useL-Glutamine ad 4mM, glucose [ Sigma)]And NAA [ Gibco ]]Feeder cells. Bispecific antibody containing cell culture supernatant was harvested by centrifugation on days 5-11 post transfection and stored at-20 ℃. General information on the recombinant expression of human immunoglobulins in, for example, HEK293 cells is given in Meissner, P.et al, Biotechnol.Bioeng (Biotechnology and bioengineering) 75(2001) 197-203.

Transient transfection in the HEK293-F System

Bispecific antibodies were generated by transient transfection of various plasmids (e.g., encoding the heavy and modified heavy chains, and the corresponding light and modified light chains) 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 the four expression plasmids and 293fectin or fectin (Invitrogen). For 2L shake flasks (Corning), 600mL HEK293-F cells were seeded at a density of 1.0E 6 cells/mL and incubated at 120rpm, 8% CO 2. The next day, cells were transfected with a mixture of ca.42ml, a) 20 mlopt-MEM (invitrogen) with 600 μ g total plasmid DNA (1 μ g/mL) encoding equimolar ratios of heavy or modified heavy chain, respectively, and corresponding light chain, at a cell density of ca.1.5e 6 cells/mL, and B) a mixture of 20mL Opti-MEM +1.2mL 293fectin or fectin (2 μ l/mL). Glucose solution was added during the fermentation process according to the glucose consumption. The supernatant containing the secreted antibody is harvested after 5-10 days, and the antibody is purified directly from the supernatant or the supernatant is frozen and stored.

Protein assay

The protein concentration of the purified antibodies and derivatives was determined by determining the Optical Density (OD) at 280nm using the molar extinction coefficient calculated based on the amino acid sequence, which was performed in accordance with Pace et al, protein science, 1995, 4, 2411-1423.

Determination of antibody concentration in supernatant

The concentration of antibodies and derivatives in cell culture supernatants was assessed by immunoprecipitation using protein a sepharose-beads (Roche). 60 μ L 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 loaded onto protein a agarose beads pre-equilibrated in TBS-NP 40. After 1h incubation at room temperature, the beads were washed 1 time with 0.5mL TBS-NP40, 2 times with 0.5mL 2XPBS (2xPBS, Roche (Roche)) and 4 times with 0.5mL 100mM Na-citrate pH 5,0 on an Ultrafree-MC-filtration column (Amicon). By adding 35. mu.l NuPAGELDS sample buffer (Invitrogen) eluted bound antibody. Half of the samples were separately compared with NuPAGEThe sample reducing agents were mixed or left unreduced and heated at 70 ℃ for 10 min. Therefore, 5-30. mu.l was applied to 4-12% NuPAGEBis-Tris SDS-PAGE (Invitrogen) (with MOPS buffer for non-reducing SDS-PAGE, and with NuPAGEMES buffer against the antioxidant running buffer additive (Invitrogen) for reduced SDS-PAGE) and stained with Coomassie blue.

The concentration of antibodies and derivatives 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 loaded onto Applied Biosystems (Applied Biosystems) Poros a/20 columns at 200mM KH2PO4, 100mM sodium citrate, pH 7.4 and eluted on Agilent (Agilent) HPLC 1100 system with 200mM NaCl, 100mM citric acid, pH 2, 5. Eluted protein was quantified by UV absorbance and peak area integration. The purified standard IgG1 antibody served as the standard.

Alternatively, the concentration of antibodies and derivatives in the cell culture supernatant is measured by sandwich-IgG-ELISA. Briefly, StreptaWell high binding streptavidin (StreptaWell high Bind streptavidin) a-96 well microtiter plates (Roche) were coated with 100 μ L/well biotinylated anti-human IgG capture molecule F (ab') 2< h-Fc γ > bi (dianova) at 0.1 μ g/mL for 1h at room temperature or alternatively overnight at 4 ℃, and then washed 3 times with 200 μ L/well PBS, 0.05% tween (PBST, Sigma (Sigma)). A dilution series of 100 μ L/well of cell culture supernatant containing the various antibodies in PBS (Sigma) was added to the wells and incubated on a microtiter plate shaker for 1-2h at room temperature. 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 1-2h with 100 μ L F (ab') 2< hfcya > pod (dianova) as detection antibody at a concentration of 0.1 μ g/mL. Unbound detection antibody was washed off in three washes with 200 μ L/well PBST and bound detection antibody was detected by addition of 100 μ LABTS/well. The determination of the absorbance was carried out on a Tecan Fluor spectrometer at a measurement wavelength of 405nm (reference wavelength 492 nm).

Protein purification

The protein was purified from the filtered cell culture supernatant with reference to standard procedures. Briefly, the antibody was loaded onto a protein a sepharose column (GE healthcare) and washed with PBS. Antibody elution was performed at pH 2.8 and the samples were immediately neutralized subsequently. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GE healthcare) in PBS or in 20mM histidine, 150mM NaCl pH 6.0. The monomeric antibody fractions are pooled, concentrated, if necessary, using, for example, a MILLIPORE Amicon Ultra (30MWCO) centrifugal concentrator, frozen at-20 ℃ or-80 ℃ and stored. Portions of the sample are provided for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, size exclusion chromatography or mass spectrometry.

SDS-PAGE

NuPAGEThe preformed gel system (Invitrogen) was used according to the manufacturer's instructions. Specifically, 10% or 4-12% NuPAGE is usedNovexBis-TRIS Prep (Pre-Cast) gel (pH6.4) and NuPAGEMES (reduced gel, with NuPAGE)Antioxidant running buffer additive) or MOPS (unreduced gel) running buffer.

Analytical size exclusion chromatography

Size exclusion chromatography to determine the aggregation and oligomerization status of the antibody was performed by HPLC chromatography. Briefly, protein A purified antibody was loaded on 300mM NaCl on an Agilent (Agilent) HPLC 1100 system, 50mM KH2PO4/K2HPO4, TosohTSKGel G3000SW column in pH 7.5 or Superdex 200 column in 2xPBS on a Dionex HPLC-system (GE healthcare). Eluted protein was quantified by integration of UV absorbance and peak area. The BioRad gel filtration Standard 151-1901 served as a standard.

Mass spectrometry

The total deglycosylation mass of the exchange antibody was determined and verified by electrospray ionization mass spectrometry (ESI-MS). Briefly, 100 μ G of purified antibody was deglycosylated with 50 mUN-glycosidase F (PNGaseF, ProZyme) at a protein concentration of at most 2mg/ml at 37 ℃ for 12-24h in 100mM KH2PO4/K2HPO4, pH 7, and subsequently desalted by HPLC on a Sephadex G25 column (GE healthcare). The mass of the various heavy and light chains was determined by ESI-MS after deglycosylation and reduction. Briefly, 50. mu.g of antibody in 115. mu.l was incubated with 60. mu.l of 1M TCEP and 50. mu.l of 8M guanidine hydrochloride and subsequently desalted. The total mass and the mass of the reduced heavy and light chains were determined by ESI-MS on a NanoMate Source equipped Q-Star Elite MS system.

IGF-1R ECD binding ELISA

The binding properties of the antibodies produced were evaluated in an ELISA assay using the extracellular domain (ECD) of IGF-1R. For this purpose, the extracellular domain of IGF-1R (residues 1-462), which includes the native leader sequence of the human IGF-IR extracellular domain of the alpha chain fused to the N-terminal His-streptavidin binding peptide-tag (His-SBP) (according to McKern et al, 1997; Ward et al, 2001) and the LI-cysteine-rich-12 domain, was cloned into pcDNA3 vector derivatives and transiently expressed in HEK293F cells. The protein sequence of IGF-1R-His-SBP ECD is shown in SEQ ID NO: 10. StreptaWell high binding streptavidin A-96 well microtiter plates (Roche) were coated with 100. mu.L/well of cell culture supernatant containing soluble IGF-1R-ECD-SBP fusion protein overnight at 4 ℃ and washed three times with 200. mu.L/well PBS, 0.05% Tween (PBST, Sigma). Subsequently, 100 μ L/well of a dilution series of each antibody in PBS (Sigma) (containing 1% BSA (fraction V, Roche) and wild type < IGF-1R > antibody as reference) and incubated at room temperature for 1-2h on a microtiter plate shaker for the dilution series, equal amounts of purified antibody were applied to the wells washed three times with 200 μ L/well PBST and bound antibody with a concentration of 0.1 μ g/mL (1: 8000) of 100 μ L/well F (ab') 2< hFc γ > pod (dianova) as detection antibody on a microtiter plate shaker at room temperature detection of 1-2h unbound detection antibody using 200 μ L/well PBST, washed off three times and bound detection antibody was detected by adding 100 μ L aban/well spectrometer on a Fluor, the determination of the absorbance was carried out at a measurement wavelength of 405nm (reference wavelength 492 nm).

IGF-1R ECD Biacore

Binding of the generated antibodies to human IGF-1R ECD was also studied by surface plasmon resonance using BIACORE T100 instrument (GE healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, goat-anti-human IgG, JIR 109-. Binding in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween 20, ph 7.4), IGF-1R ECD (R & D System or internally purified) was added to the solution at various concentrations association was measured by IGF-1R ECD injection for 80 seconds-3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3-10 minutes and KD values were assessed using the 1: 1 Langmuir binding model (Langmuir binding model.) monovalent IGF-1R ECD binding was obtained due to the low loading density and capture level of the < IGF-1R > antibody, negative control data (e.g. buffer curve) were subtracted from the sample curves, for correcting the baseline drift inherent to the system and for noise signal reduction the Biacore T100 evaluation software version 1.1.1 was used to analyze the S-curves (sensorgrams) and for calculating the affinity data fig. 11 shows a Biacore assay summary.

Example 1:

monospecific bivalent < IGF-1R > antibodies were prepared, expressed, purified and characterized, wherein the variable domains CL and CH1 were replaced with each other (abbreviated herein as < IGF-1R > CL-CH1 exchange antibody).

Example 1A

Preparation of expression plasmids for monospecific bivalent < IGF-1R > CL-CH1 exchange antibodies

The sequences of the heavy and light chain variable domains of the monospecific bivalent < IGF-1R > CL-CH1 exchange antibody including various leader sequences described in this example were derived from human < IGF-1R > antibody heavy chain (SEQ ID NO: 1, plasmid 4843-pUC-HC-IGF-1R) and light chain (SEQ ID NO: 2, plasmid 4842-pUC-LC-IGF-1R) as described in WO 2005/005635, and the heavy and light chain constant domains were derived from human antibodies (C-. kappa.and IgG 1).

Will code<IGF-1R>Antibody leader sequences, heavy chain variable domains (VH) and gene segments of the human kappa-light chain domain (CL) are linked and fused to the 5' end of the Fc domain of the human gamma 1-heavy chain constant domain (hinge-CH 2-CH 3). DNA encoding various fusion proteins obtained by exchanging the CH1 domain with the CL domain (CH1-CL exchange) was produced by gene synthesis and is hereinafter represented as<IGF-1R>HC^**(SEQ ID NO：3)。

<IGF-1R>The gene segments of the antibody leader sequence, light chain variable domain (VL) and human γ 1-heavy chain constant domain (CH1) are linked as separate chains. DNA encoding various fusion proteins obtained by exchanging the CL domain with the CH1 domain (CL-CH1 exchange) was produced by gene synthesis and is hereinafter represented as<IGF-1R>LC^**(SEQ ID NO：4)。

FIGS. 5 and 6 show modifications<IGF-1R>HC^**Heavy chain and modified<IGF-1R>LC^**Schematic representation of the protein sequence of the light chain.

In the following, various expression vectors are briefly described:

vector pUC-HC^**-IGF-1R

Vector pUC-HC^**IGF-1R is, for example, for transient expression of the CL-CH1 exchange in HEK293(EBNA) cells<IGF-1R>Heavy chain HC^**(expression cassette constructed from cDNA; with CMV-intron A) or expression plasmids for stable expression in CHO cells.

Removing device<IGF-1R>HC^**In addition to the expression cassette, the vector comprises:

an origin of replication from the vector pUC18, which allows replication of the plasmid in E.coli, and

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

<IGF-1R>HC^**The transcriptional unit of a gene consists of the following elements:

AscI restriction site at the 5' terminus

Immediate early enhancer and promoter from human cytomegalovirus,

-a subsequent intron A sequence,

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

-an immunoglobulin light chain signal sequence,

-a person<IGF-1R>Mature HC^**Chains encoding a fusion of a human heavy chain variable domain (VH) and a human kappa-light chain constant domain (CL) fused to the 5' end of the Fc domain of a human gamma 1-heavy chain constant domain (hinge-CH 2-CH3)

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

the restriction site SgrAI at the 3' end.

Heavy chain^**CL-CH1 exchange<IGF-1R>HC^**Expression vector pUC-HC^**The plasmid map of IGF-1R is shown in FIG. 7.<IGF-1R>HC^**(including the signal sequence) is as set forth in SEQ ID NO: 3.

Vector pUC-LC^**-IGF-1R

Vector pUC-LC^**IGF-1R is, for example, for transient expression of the CL-CH1 exchange in HEK293(EBNA) cells<IGF-1R>Light chain LC^**(expression cassette constructed from cDNA; with CMV-intron A) or expression plasmids for stable expression in CHO cells.

Removing device<IGF-1R>LC^**In addition to the expression cassette, the vector comprises:

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

<IGF-1R>LC^**The transcriptional unit of a gene consists of the following elements:

the restriction site Sse8387I at the 5' end

Immediate early enhancer and promoter from human cytomegalovirus,

-a subsequent intron A sequence,

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

an immunoglobulin heavy chain signal sequence,

-a person<IGF-1R>Antibody maturation LC^**Chains encoding a fusion of a human light chain variable domain (VL) and a human gamma 1-heavy chain constant domain (CH1)

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

the restriction sites SalI and FseI at the 3' end.

Light chain^**CL-CH1 exchange<IGF-1R>LC^**Expression vector pUC-LC^**The plasmid map of IGF-1R is shown in FIG. 8.<IGF-1R>LC^**(including the signal sequence) is as set forth in SEQ ID NO: 4 is provided.

Plasmid pUC-HC^**IGF-1R and pUC-LC^**IGF-1R can be used for transient or stable co-transfection into, for example, HEK293EBNA or CHO cells (2-vector systems). For comparative reasons, wild type<IGF-1R>The antibody was transiently expressed by plasmids 4842-pUC-LC-IGF-1R (SEQ ID NO: 2) and 4843-pUC-HC-IGF-1R (SEQ ID NO: 1) similar to those described in this example.

To obtain higher expression levels of transient expression in HEK293EBNA cells, one would like to use<IGF-1R>HC^**The expression cassette was subcloned via AscI, SgrAI sites and<IGF-1R>LC^**the expression cassette was subcloned into the FseI site via Sse8387IIn a 4700pUC-Hyg _ OriP expression vector comprising:

an OriP element, and

-a hygromycin resistance gene as a detectable marker.

The heavy and light chain transcription units can be subcloned into 2 independent 4700-pUC-Hyg-OriP vectors for co-transfection (2-vector system), or they can be cloned into one common 4700-pUC-Hyg-OriP vector (1-vector system) for subsequent transient or stable transfection with the resulting vector. FIG. 9 shows a plasmid map of the basic vector 4700-pUC-OriP.

Example 1B

Preparation of monospecific bivalent < IGF-1R > CL-CH1 exchange antibody expression plasmid

Assembly of nucleic acid segments including wild-type by ligation of corresponding nucleic acid segments using known recombinant methods and techniques<IGF-1R>Of exchanged Fab sequences of antibodies<IGF-1R>Fusion gene (HC)^**And LC^**Fusion gene).

Encoding IGF-1R HC^**And LC^**The nucleic acid sequences of (a) were each synthesized by chemical synthesis and subsequently cloned into a pPCRscript (Stratagene) -based pGA4 cloning vector in Geneart (Regensburg, Germany). Will encode IGF-1R HC^*Is ligated into various E.coli plasmids via PvuII and BmgBI restriction sites to generate the final vector pUC-HC^**-IGF-1R; will encode various IGF-1R LCs^*Into various E.coli plasmids via PvuII and SalI restriction sites to generate the final vector pUC-LC^**-IGF-1R. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient and stable transfection, larger quantities of plasmid (Nucleobond AX, Macherey-Nagel) were prepared by plasmid preparations from transformed E.coli cultures.

Example 1C

Transient expression of monospecific bivalent < IGF-1R > CL-CH1 exchange antibody, purification and confirmation of identity by mass spectrometry

Recombination<IGF-1R>CL-CH1 exchange antibody was generated by transient co-transfection of plasmid pUC-HC in HEK293-F suspension cells^**IGF-1R and pUC-LC^**IGF-1R, as described above.

The expressed and secreted monospecific bivalent < IGF-1R > CL-CH1 exchange antibody was purified from the filtered cell culture supernatant by corresponding protein a affinity chromatography as described above. Briefly, cell culture supernatants from transient transfections containing < IGF-1R > CL-CH1 exchange antibody were clarified by centrifugation and filtration and loaded on a protein a hitrap select Xtra column (GE healthcare) equilibrated with PBS buffer (10mM Na2HPO4, 1mM kh2PO4, 137mM NaCl and 2.7mM KCl, pH 7.4). Unbound protein was washed out with PBS equilibration buffer followed by 0.1M sodium citrate buffer, pH 5.5 and washed with PBS. Elution of the antibody was achieved with 100mM sodium citrate, pH 2,8, followed immediately by neutralization of the sample with 300. mu.l of 2M TrispH 9.0/2ml fraction. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography on a HiLoad 26/60 Superdex 200 preparative column (GE healthcare) in 20mM histidine, 150mM NaCl pH 6.0, and the monomeric antibody fractions were subsequently concentrated using a MILLIPORE Amicon-15 centrifugal concentrator. (ii) freezing and storing < IGF-1R > CL-CH1 exchange antibody at-20 ℃ or-80 ℃. The integrity of the < IGF-1R > CL-CH1 exchanged antibody was analyzed by SDS-PAGE in the presence and absence of reducing agents and subsequent staining with Coomassie Brilliant blue, as described above. The monomeric status of the IGF-1R > CL-CH1 exchanged antibody was confirmed by analytical size exclusion chromatography. (FIG. 12) the characterized samples were provided for subsequent protein analysis and functional characterization. ESI mass spectrometry confirmed the theoretical molecular weight of the fully deglycosylated < IGF-1R > CL-CH1 exchange antibody.

Example 1D

Analysis of IGF-1R binding characteristics of monospecific bivalent < IGF-1R > CL-CH1 exchange antibodies in IGF-1R ECD binding ELISA and by Biacore

The binding properties of monospecific bivalent < IGF-1R > CL-CH1 exchange antibodies were evaluated in an ELISA assay using the IGF-1R extracellular domain (ECD) as described above. For this purpose, the extracellular domain of IGF-1R (residues 1-462, which includes the native leader sequence of the human IGF-IR extracellular domain of the alpha chain fused to the N-terminal His-streptavidin binding peptide-tag (His-SBP) (according to McKern et al, 1997; Ward et al, 2001) and the LI-cysteine-rich-12 domain) was cloned into pcDNA3 vector derivatives and transiently expressed in HEK293F cells. The protein sequence of IGF-1R-His-SBPECD is given above. The titration curves obtained showed that the < IGF-1R > CL-CH1 exchange antibody was functional and showed comparable binding properties and kinetics to the wild-type < IGF-1R > antibody within the error of the method, and thus appeared to be fully functional (fig. 13).

These findings are corroborated by Biacore data on various purified antibodies, which showed that the monospecific bivalent < IGF-1R > CL-CH1 exchange antibody (with a KD of 3.7pM) had an affinity and binding kinetics for IGF-1R ECD comparable to the original wild-type < IGF-1R > antibody (with a KD of 3.2 pM).

Example 1G

Analysis of IGF-1R binding characteristics of monospecific bivalent < IGF-1R > CL-CH1 exchange antibodies by FACS Using I24 cells overexpressing IGF-1R

To validate, the binding activity of < IGF-1R > CL-CH1 exchange antibody to IGF-1R overexpressed on the surface of I24 cells (NIH 3T3 cells expressing recombinant human IGF-1R, Roche) was studied by FACS. Briefly, 5x10E 5I 24 cells/FACS tubes were incubated with dilutions of purified < IGF-1R > CL-CH1 exchange antibody and wild type < IGF-1R > antibody as reference and incubated for 1h on ice. Unbound antibody was washed away with 4ml ice-cold pbs (gibco) + 2% fcs (gibco). Subsequently, the cells were centrifuged (5min, 400g) and bound antibody was detected with F (ab') 2< hFc γ > PE conjugate (Dianova) on ice for 1h under dark conditions. Unbound detection antibody was washed away with 4ml ice-cold PBS (Gibco) + 2% FCS (Gibco). Subsequently, the cells were centrifuged (5min, 400g), resuspended in 300-500 μ L PBS, and bound detection antibody was quantified on FACSCalibur or FACS Canto (BD (FL2 channel, 10.000 cells/harvest) < IGF-1R > CL-CH1 exchange antibody and wild type < IGF-1R > reference antibody binding to IGF-1R on I24 cells resulted in comparable concentration-dependent changes in mean fluorescence intensity during the experiment, including respective isotype controls to exclude any non-specific binding events.

Example 2:

description of monospecific bivalent < ANGPT2> wild-type antibody

Example 2A

Preparation of expression plasmid for monospecific bivalent < ANGPT2> wild-type antibody

The sequences of the heavy and light chain variable domains of the monospecific bivalent ANGPT2< ANGPT2> wild-type antibody described in this example, including various leader sequences, were derived from the heavy (SEQ ID NO: 6) and light (SEQ ID NO: 7) chains of the human < ANGPT2> antibody as described in WO2006/045049, and the heavy and light chain constant domains were derived from human antibodies (C- κ and IgG 1).

The wild-type < ANGPT2> antibody was cloned into plasmids SB04-pUC-HC-ANGPT2(SEQ ID NO: 6) and SB06-pUC-LC-ANGPT2(SEQ ID NO: 7) similar to the vector described in the previous example 1A.

For comparative reasons and for co-expression experiments (see example 3), the wild-type < ANGPT2> antibody was transiently (co-) expressed from plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT 2.

Example 2B

The nucleic acid sequences encoding ANGPT2> HC and LC, respectively, were synthesized by chemical synthesis and subsequently cloned into pcrscript (stratagene) -based pGA4 cloning vector in Geneart (Regensburg, germany). Cloning the expression cassettes encoding < ANGPT2> HC into the respective E.coli plasmids to generate the final vector SB04-pUC-HC-ANGPT 2; the expression cassettes encoding the various < ANGPT2> LCs were cloned into the respective E.coli plasmids to generate the final vector SB06-pUC-LC-ANGPT 2. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient and stable transfection, larger quantities of plasmid (Nucleobond AX, Macherey-Nagel) were prepared by plasmid preparations from transformed E.coli cultures.

Example 3

A bispecific bivalent < ANGPT2-IGF-1R > antibody was expressed in which the constant domains CL and CH1 are replaced with each other in the heavy and light chains that specifically bind IGF-1R (abbreviated herein as < ANGPT2-IGF-1R > CL-CH1 exchange antibody).

Example 3A

Transient co-expression and purification of < IGF-1R > CL-CH1 exchange antibody and < ANGPT2> wild-type antibody in HEK293EBNA cells to generate bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody

For generation through a channel located at one side<IGF-1R>The CL-CH1 exchange antibody Fab recognizes IGF-1R and passes through the other side<ANGPT2>Wild type Fab region identification<ANGPT2>2 encode<IGF-1R>Expression plasmids of the CL-CH1 crossover antibody (example 1A) with 2 plasmids encoding<ANGPT2>Expression plasmids for wild-type antibodies (example 2A) were co-expressed. It is hypothesized that wild-type heavy chain HC and CL-CH1 exchange for heavy chain HC^**Statistical association, which leads to bispecific bivalency<IGF-1R-ANGPT2>Generation of CL-CH1 exchange antibody. Under the assumption that both antibodies are equally well expressed and no by-products are considered, this should result in three major products in a 1: 2: 1 ratio: A)<IGF-1R>CL-CH1 exchange antibody, B) bispecific<IGF-1R-ANGPT2>CL-CH1 exchange antibody and C)<ANGPT2>A wild-type antibody. Several by-products can be expected. However, since only the CL-CH1 domain is exchanged, by-productsThe frequency should be reduced compared to the intact Fab exchange type. Please note that because<ANGPT2>Wild type antibody display ratio<IGF-1R>Wild type and<IGF-1R>higher expression transient expression yield of CL-CH1 exchanged antibody, therefore<ANGPT2>Wild type antibody plasmids and<IGF-1R>the ratio of CL-CH1 crossover antibody plasmid was oriented to favor expression<ANGPT2>The orientation of the wild-type antibody is off set.

To form the main product A)<IGF-1R>CL-CH1 exchange antibody, B) bispecific<ANGPT2-IGF-1R>CL-CH1 exchange antibody and C)<ANGPT2>Mixture of wild-type antibodies, four plasmids pUC-HC were co-transfected in HEK293-F cells suspended as described above^**IGF-1R and pUC-LC^**IGF-1R and plasmids SB04-pUC-HC-ANGPT2 and SB06-pUC-LC-ANGPT 2. The captured supernatant contains the main product A)<IGF-1R>CL-CH1 exchange antibody, B) bispecific<ANGPT2-IGF-1R>CL-CH1 exchange antibody and C)<ANGPT2>A mixture of wild-type antibodies and is denoted as "bispecific CL-CH1 exchange mixture". The cell culture supernatant containing the bispecific CL-CH1 exchange mixture was captured by centrifugation and subsequently purified as described above. FIG. 14

The integrity of the antibody mixture was analyzed by SDS-PAGE in the presence and absence of reducing agent and subsequent staining with Coomassie Brilliant blue, as described above. SDS-PAGE showed the presence of 2 different heavy and light chains in the preparation as expected (reduced gel). The monomeric state of the antibody mixture was verified by analytical size exclusion chromatography and showed that the purified antibody species was in the monomeric state. The characterized sample is provided for subsequent protein analysis and functional characterization.

Example 3B

Detection of functional bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody in cellular FACS bridging assay against I24 IGF-1R expressing cells

To confirm the presence of functional bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody in the purified bispecific CL-CH1 exchange mixture from transient co-expressed major product a) < IGF-1R > CL-CH1 exchange antibody described in example 3A, B) bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody and C) < ANGPT2> wild-type antibody, cell facscif IGF-1R-ANGPT2 bridging assays were performed on I24 cells (NIH 3T3 cells expressing recombinant human IGF-1R, Roche). The principle of this assay is depicted in fig. 10. The bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody present in the purified antibody mixture was capable of simultaneously binding to IGF-1R in I24 cells and binding to ANGPT 2; and thus will bridge its two target antigens with two opposing Fab regions.

Briefly, 5x10E 5I 24 cells/FACS tubes were incubated with the entire purified antibody mixture and incubated on ice for 1h (titration of 160 μ g/ml mixture). Various purified antibodies wild-type < IGF-1R > and < ANGPT2> were applied to I24 cells as controls. Unbound antibody was washed with 4mL ice cold PBS (Gibco) + 2% FCS (Gibco), cells were centrifuged (5min, 400g) and bound bispecific antibody was detected on ice with 50 μ l of 2 μ g/mL human ANGPT2(R & D Systems)) for 1 h. Subsequently, unbound ANGPT2 was washed once or twice with 4mL ice-cold pbs (gibco) + 2% fcs (gibco), the cells were centrifuged (5min, 400g) and bound ANGPT2 was detected on ice for 45 min with 50 μ l of 5 μ g/mL < ANGPT2> mIgG 1-biotin antibody (BAM0981, R & D Systems (R & D Systems)); alternatively, cells were incubated with 50 μ l of 5 μ g/mL mIgG 1-biotin-isotype control (R & D Systems). Unbound detection antibody was washed with 4ml ice-cold PBS (Gibco) + 2% FCS (Gibco), the cells were centrifuged (5min, 400g) and bound detection antibody was detected on ice with 50. mu.l of 1: 400 streptavidin-PE conjugate (Invitrogen/Zymed) for 45 min under dark conditions. Unbound streptavidin-PE conjugate was washed away with 4ml ice-cold PBS (Gibco) + 2% FCS (Gibco). Subsequently, the cells were centrifuged (5min, 400g), resuspended in 300-500 μ L PBS, and the bound streptavidin-PE conjugate quantified on FACSCalibur (BD (FL2 channel, 10.000 cells/harvest.) during the experiment, respective isotype controls were included to exclude any non-specific binding events.

The results in figure 15 show that incubation of purified antibody-exchanged mixtures (< ANGPT2-IGF-1R > CL-CH1 exchanged antibody) with co-expression from exchanged antibodies (< IGF-1R > CL-CH1 exchanged antibody) and wild-type antibodies (< ANGPT2> wild-type antibody) resulted in a significant shift in fluorescence, indicating the presence of a functional bispecific < ANGPT2-IGF-1R > CL-CH1 exchanged antibody capable of simultaneously binding to IGF-1R in I24 cells and binding to ANGPT 2; and thus bridges its two target antigens using two opposing Fab regions. In contrast, the respective < IGF-1R > and < Ang-2> control antibodies did not cause a shift in fluorescence in the FACS bridging assay.

In summary, these data show that: respective wild-type and exchanged plasmid functional bispecific antibodies can be generated by co-expression. The yield of the correct bispecific antibody can be increased by forcing the correct heterodimerization of the wild-type and modified exchange heavy chains, for example, using bulge-entry-hole technology and disulfide bond stabilization (see example 4).

Example 4

Expression of bivalent bispecific < ANGPT2-IGF-1R > CL-CH1 exchange antibody with modified CH3 Domain (bulge-entry-hole)

To further improve the dual specificity<ANGPT2-IGF-1R>Yield of CL-CH1 exchange antibody, applying the bulge-in-hole technique to<IGF-1R>CL-CH1 exchange and wild type<ANGPT2>Co-expression of antibodies to obtain a homogeneous and functional bispecific antibody preparation. For the purpose of this purpose,<IGF-1R>heavy chain of CL-CH1 exchange antibody^*HC^*The CH3 domain in (a) is replaced with SEQ ID NO: domain CH3 (bulge) of 8 and wild type<ANGPT2>The CH3 domain in the heavy chain of the antibody was replaced with SEQ ID NO: the CH3 domain (pore) of 9, or vice versa. In addition, disulfide bonds may be included to increase stability and yield as well as additional residues to form ionic bridges and increase heterodimerization yield (EP 1870459a 1).

Transient co-expression and purification of the thus generated bivalent bispecific ANGPT2-IGF-1R > CL-CH1 exchange antibody with a modified CH3 domain (bulge-entry-hole) was performed as described in example 3.

It should be noted that the optimization of heterodimerization can be achieved, for example, as follows: by using different bulge-entry-hole techniques, such as introducing additional disulfide bonds into the CH3 domain, e.g. introducing Y349C into the "bulge chain" and D356C into the "hole chain", and/or combining to use residue R409D for bulge residues as described in EP 1870459a 1; K370E (K409D) and use of D399K for the hole residues; E357K.

Similar to example 4, other bivalent, bispecific CH1-CL exchange antibodies with modified CH3 domains (bulge-entry-hole) can be prepared that exchange heavy and light chains against ANGPT2 and another target antigen (using the aforementioned ANGPT2 heavy and light chains and CH1-CL of an antibody against the other target^**HC^**And LC^**Whereby both heavy chains are modified by "bulge-entry-hole"), or against IGF-1R and another target (exchange of heavy and light chains with the heavy and light chains of an antibody against said other target and CH1-CL described above^**HC^**And LC^**Whereby both heavy chains are modified by "bulge-entry-hole").

Sequence listing

<110> Hoffman-Ravigh Co., Ltd

<120> bivalent bispecific antibody

<130>24679 EP

<150>EP 07024865

<151>2007-12-21

<160>10

<170>PatentIn version 3.2

<210>1

<211>467

<212>PRT

<213> Artificial

<220>

<223> amino acid sequence of wild type < IGF-1R > antibody heavy chain

<400>1

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ile Ile Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Phe Cys Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly

115 120 125

Arg Gly Thr Leu Val Ser Val Ser Ser Ala Ser Thr Lys Gly Pro Ser

130 135 140

Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala

145 150 155 160

Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val

165 170 175

Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala

180 185 190

Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val

195 200 205

Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His

210 215 220

Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230 235 240

Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly

245 250 255

Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met

260 265 270

Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His

275 280 285

Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val

290 295 300

His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr

305 310 315 320

Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly

325 330 335

Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro A la Pro Ile

340 345 350

Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val

355 360 365

Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser

370 375 380

Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu

385 390 395 400

Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro

405 410 415

Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val

420 425 430

Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met

435 440 445

His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser

450 455 460

Pro Gly Lys

465

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<223> amino acid sequence of wild-type < IGF-1R > antibody light chain

<400>2

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

100 105 110

Lys Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys

115 120 125

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

130 135 140

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

145 150 155 160

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn A la Leu Gln

165 170 175

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

180 185 190

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

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Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

210 215 220

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

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<223> < IGF-1R > CL-CH1 amino acid sequence of heavy chain of the exchange antibody,

wherein the heavy chain domain CH1 is replaced by a light chain domain CL

<400>3

Met Glu Phe Gly Leu Ser Trp Val Phe Leu Val Ala Leu Leu Arg Gly

1 5 10 15

Val Gln Cys Gln Val Glu Leu Val Glu Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Gln Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ile Ile Trp Phe Asp Gly Ser Ser Thr Tyr Tyr Ala

65 70 75 80

Asp Ser Val Arg Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Phe Cys Ala Arg Glu Leu Gly Arg Arg Tyr Phe Asp Leu Trp Gly

115 120 125

Arg Gly Thr Leu Val Ser Val Ser Ser Ala Ser Val Ala Ala Pro Ser

130 135 140

Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala

145 150 155 160

Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val

165 170 175

Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser

180 185 190

Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr

195 200 205

Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys

210 215 220

Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn

225 230 235 240

Arg Gly Glu Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro A la Pro

245 250 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys

465 470

<210>4

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<223> < IGF-1R > CL-CH1 amino acid sequence of Light Chain (LC) of the crossover antibody,

wherein the light chain domain CL is replaced by the heavy chain domain CH 1.

<400>4

Met Glu Ala Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Trp Leu Pro

1 5 10 15

Asp Thr Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser

20 25 30

Leu Ser Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser

35 40 45

Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro

50 55 60

Arg Leu Leu Ile Tyr Asp Ala Ser Lys Arg Ala Thr Gly Ile Pro Ala

65 70 75 80

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser

85 90 95

Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser

100 105 110

Lys Trp Pro Pro Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ser Lys

115 120 125

Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser

130 135 140

Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys

145 150 155 160

Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly A la Leu

165 170 175

Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu

180 185 190

Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr

195 200 205

Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val

210 215 220

Asp Lys Lys Val Glu Pro Lys Ser Cys

225 230

<210>5

<211>557

<212>PRT

<213> Artificial

<220>

<223> amino acid sequence of IGF-1R extracellular domain His-streptavidin binding peptide-tag (IGF-1R-His-SBP ECD)

<400>5

Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu

1 5 10 15

Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile

20 25 30

Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg

35 40 45

Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu Leu Ile

50 55 60

Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val

65 70 75 80

Ile Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu

85 90 95

Gly Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Trp Lys Leu Phe

100 105 110

Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile

115 120 125

Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly Ala Ile Arg Ile Glu

130 135 140

Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile

145 150 155 160

Leu Asp Ala Val Ser Asn Asn Tyr Ile Val Gly Asn Lys Pro Pro Lys

165 170 175

Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys

180 185 190

Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr

195 200 205

Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys

210 215 220

Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser

225 230 235 240

Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr

245 250 255

Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu

260 265 270

Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala

275 280 285

Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met

290 295 300

Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr

305 310 315 320

Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys

325 330 335

Lys Thr Lys Thr Ile Asp Ser Val Thr Ser Ala Gln Met Leu Gln Gly

340 345 350

Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn

355 360 365

Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val

370 375 380

Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser

385 390 395 400

Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly

405 410 415

Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp

420 425 430

Asp Trp Asp His Arg Asn Leu Thr Ile Lys Ala Gly Lys Met Tyr Phe

435 440 445

Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu

450 455 460

Val Thr Gly Thr Lys Gly Arg Gln Ser Lys Gly Asp Ile Asn Thr Arg

465 470 475 480

Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Ala Ala Ala Leu

485 490 495

Glu Val Leu Phe Gln Gly Pro Gly Thr His His His His His His Ser

500 505 510

Gly Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly

515 520 525

Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro

530 535 540

Gln Gly Gln Arg Glu Pro Ser Gly Gly Cys Lys Leu Gly

545 550 555

<210>6

<211>471

<212>PRT

<213> Artificial

<220>

<223> amino acid sequence of wild type angiogenin-2 < ANGPT2> antibody heavy chain

<400>6

Met Glu Leu Gly Leu Ser Trp Val Phe Leu Val Ala Ile Leu Glu Gly

1 5 10 15

Val Gln Cys Glu Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe

35 40 45

Ser Ser Tyr Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ser Tyr Ile Ser Ser Ser Gly Ser Thr Ile Tyr Tyr Ala

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn

85 90 95

Ser Leu Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Asp Leu Leu Asp Tyr Asp Ile Leu Thr Gly Tyr

115 120 125

Gly Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr

130 135 140

Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser

145 150 155 160

Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu

165 170 175

Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His

180 185 190

Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser

195 200 205

Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys

210 215 220

Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu

225 230 235 240

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

245 250 255

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

260 265 270

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

275 280 285

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

290 295 300

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

305 310 315 320

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

325 330 335

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

340 345 350

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

355 360 365

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

370 375 380

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

385 390 395 400

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

405 410 415

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

420 425 430

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

435 440 445

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

450 455 460

Leu Ser Leu Ser Pro Gly Lys

465 470

<210>7

<211>219

<212>PRT

<213> Artificial

<220>

<223> amino acid sequence of wild type angiogenin-2 < ANGPT2> antibody light chain

<400>7

Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro Gly

1 5 10 15

Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu His Ser

20 25 30

Asn Gly Tyr Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro Gly Gln Ser

35 40 45

Pro Gln Leu Leu Ile Tyr Leu Gly Ser Asn Arg Ala Ser Gly Val Pro

50 55 60

Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile

65 70 75 80

Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Gly

85 90 95

Thr His Trp Pro Pro Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys

100 105 110

Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu

115 120 125

Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe

130 135 140

Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn A la Leu Gln

145 150 155 160

Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser

165 170 175

Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu

180 185 190

Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser

195 200 205

Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215

<210>8

<211>107

<212>PRT

<213> Artificial

<220>

<223> amino acid sequence of CH3 domain with T366W exchange (bulge) for use in bulge-entry-hole technology

<400>8

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu

1 5 10 15

Glu Met Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210>9

<211>107

<212>PRT

<213> Artificial

<220>

<223> CH3 domain with T366W exchange (bulge) for use in bulge-in-hole technology

Amino acid sequence of (1)

<400>9

Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp

1 5 10 15

Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe

20 25 30

Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu

35 40 45

Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe

50 55 60

Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly

65 70 75 80

Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr

85 90 95

Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

100 105

<210>10

<211>557

<212>PRT

<213> Artificial

<220>

<223> IGF-1R extracellular domain His-streptavidin binding peptide-tag (IGF-1R-His-SBP ECD)

Amino acid sequence of (1)

<400>10

Met Lys Ser Gly Ser Gly Gly Gly Ser Pro Thr Ser Leu Trp Gly Leu

1 5 10 15

Leu Phe Leu Ser Ala Ala Leu Ser Leu Trp Pro Thr Ser Gly Glu Ile

20 25 30

Cys Gly Pro Gly Ile Asp Ile Arg Asn Asp Tyr Gln Gln Leu Lys Arg

35 40 45

Leu Glu Asn Cys Thr Val Ile Glu Gly Tyr Leu His Ile Leu Leu Ile

50 55 60

Ser Lys Ala Glu Asp Tyr Arg Ser Tyr Arg Phe Pro Lys Leu Thr Val

65 70 75 80

Ile Thr Glu Tyr Leu Leu Leu Phe Arg Val Ala Gly Leu Glu Ser Leu

85 90 95

Gly Asp Leu Phe Pro Asn Leu Thr Val Ile Arg Gly Trp Lys Leu Phe

100 105 110

Tyr Asn Tyr Ala Leu Val Ile Phe Glu Met Thr Asn Leu Lys Asp Ile

115 120 125

Gly Leu Tyr Asn Leu Arg Asn Ile Thr Arg Gly A la Ile Arg Ile Glu

130 135 140

Lys Asn Ala Asp Leu Cys Tyr Leu Ser Thr Val Asp Trp Ser Leu Ile

145 150 155 160

Leu Asp Ala Val Ser Asn Asn Tyr Ile Val Gly Asn Lys Pro Pro Lys

165 170 175

Glu Cys Gly Asp Leu Cys Pro Gly Thr Met Glu Glu Lys Pro Met Cys

180 185 190

Glu Lys Thr Thr Ile Asn Asn Glu Tyr Asn Tyr Arg Cys Trp Thr Thr

195 200 205

Asn Arg Cys Gln Lys Met Cys Pro Ser Thr Cys Gly Lys Arg Ala Cys

210 215 220

Thr Glu Asn Asn Glu Cys Cys His Pro Glu Cys Leu Gly Ser Cys Ser

225 230 235 240

Ala Pro Asp Asn Asp Thr Ala Cys Val Ala Cys Arg His Tyr Tyr Tyr

245 250 255

Ala Gly Val Cys Val Pro Ala Cys Pro Pro Asn Thr Tyr Arg Phe Glu

260 265 270

Gly Trp Arg Cys Val Asp Arg Asp Phe Cys Ala Asn Ile Leu Ser Ala

275 280 285

Glu Ser Ser Asp Ser Glu Gly Phe Val Ile His Asp Gly Glu Cys Met

290 295 300

Gln Glu Cys Pro Ser Gly Phe Ile Arg Asn Gly Ser Gln Ser Met Tyr

305 310 315 320

Cys Ile Pro Cys Glu Gly Pro Cys Pro Lys Val Cys Glu Glu Glu Lys

325 330 335

Lys Thr Lys Thr Ile Asp Ser Val Thr Ser Ala Gln Met Leu Gln Gly

340 345 350

Cys Thr Ile Phe Lys Gly Asn Leu Leu Ile Asn Ile Arg Arg Gly Asn

355 360 365

Asn Ile Ala Ser Glu Leu Glu Asn Phe Met Gly Leu Ile Glu Val Val

370 375 380

Thr Gly Tyr Val Lys Ile Arg His Ser His Ala Leu Val Ser Leu Ser

385 390 395 400

Phe Leu Lys Asn Leu Arg Leu Ile Leu Gly Glu Glu Gln Leu Glu Gly

405 410 415

Asn Tyr Ser Phe Tyr Val Leu Asp Asn Gln Asn Leu Gln Gln Leu Trp

420 425 430

Asp Trp Asp His Arg Asn Leu Thr Ile Lys Ala Gly Lys Met Tyr Phe

435 440 445

Ala Phe Asn Pro Lys Leu Cys Val Ser Glu Ile Tyr Arg Met Glu Glu

450 455 460

Val Thr Gly Thr Lys Gly Arg Gln Ser Lys Gly Asp Ile Asn Thr Arg

465 470 475 480

Asn Asn Gly Glu Arg Ala Ser Cys Glu Ser Asp Val Ala Ala Ala Leu

485 490 495

Glu Val Leu Phe Gln Gly Pro Gly Thr His His His His His His Ser

500 505 510

Gly Asp Glu Lys Thr Thr Gly Trp Arg Gly Gly His Val Val Glu Gly

515 520 525

Leu Ala Gly Glu Leu Glu Gln Leu Arg Ala Arg Leu Glu His His Pro

530 535 540

Gln Gly Gln Arg Glu Pro Ser Gly Gly Cys Lys Leu Gly

545 550 555

Claims

1. A bivalent, bispecific antibody comprising:

2. Antibody according to claim 1, characterized in that

The CH3 domain of one heavy chain and the CH3 domain of the other heavy chain are in contact at an interface that includes the initial interface between the antibody CH3 domains;

wherein the interface is altered to facilitate formation of the bivalent, bispecific antibody, wherein the alteration is characterized by:

a) the CH3 domain of one heavy chain is altered,

the amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby generating a bulge within the interface of the CH3 domain of one heavy chain that can be positioned in a cavity within the interface of the CH3 domain of the other heavy chain

And is

b) The CH3 domain of the other heavy chain is altered,

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

3. Antibody according to claim 2, characterized in that

The amino acid residue having a larger side chain volume is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W).

4. Antibody according to any one of claims 2 or 3, characterized in that

The amino acid residue having a smaller side chain volume is selected from the group consisting of alanine (a), serine (S), threonine (T), valine (V).

5. Antibody according to any one of claims 2 to 3, characterized in that

The two CH3 domains were further altered by introducing cysteine (C) as an amino acid at the corresponding position of each CH3 domain.

6. Antibody according to claim 4, characterized in that

7. Antibody according to claim 1, characterized in that

One of the constant heavy domain CH3 of both heavy chains is replaced by the constant heavy domain CH 1; and the other constant heavy chain domain CH3 was replaced with a constant light chain domain CL.

8. A process for the preparation of a bivalent, bispecific antibody according to claim 1, comprising the following steps:

a) the host cell is transformed with the following items,

-a vector comprising nucleic acid molecules encoding the light and heavy chains of an antibody that specifically binds to a first antigen, and

c) recovering the antibody molecule from the culture.

9. A host cell comprising

A vector comprising nucleic acid molecules encoding the light and heavy chains of an antibody that specifically binds to a first antigen,

10. A composition of bivalent bispecific antibody according to claims 1 to 7.

11. The composition according to claim 10, wherein the composition is a pharmaceutical or diagnostic composition, wherein at least one of the two different antigens specifically bound by the bivalent, bispecific antibody is a tumor antigen.

12. A pharmaceutical composition comprising a bivalent bispecific antibody according to claims 1 to 7 and at least one pharmaceutically acceptable excipient, wherein at least one of the two different antigens to which the bivalent bispecific antibody specifically binds is a tumor antigen.