HK1170495B

HK1170495B - Tri- or tetraspecific antibodies

Info

Publication number: HK1170495B
Application number: HK12111052.8A
Authority: HK
Inventors: Rebecca Croasdale; Christian Klein; Wolfgang Schaefer; Juergen Michael Schanzer
Original assignee: 霍夫曼-拉罗奇有限公司
Priority date: 2009-05-27
Filing date: 2010-05-25
Publication date: 2016-04-29

Description

Tri-or tetraspecific antibodies

The present invention relates to novel tri-or tetra-specific antibodies, their preparation and use.

Background

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

A wide variety of recombinant multispecific 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.15(1997) 159-1234; WO 2001/077342; and Morrison, S.L., Nature Biotech.25(2007) 1233-1234).

Also, several other new formats such as anti-, tri-or tetra-antibodies, miniantibodies, several single chain formats (scFv, Bis-scFv) capable of binding two or more antigens, no longer retaining the core structure of the antibody (IgA, IgD, IgE, IgG or IgM), have been developed (Holliger, P., et. al, Nature Biotech.23(2005) 1126-1296; Fischer, N., and Leger, O., Pathiology 74(2007) 3-14; Shen, J., et. al., J. Methol. methods (318) 2007-74; Wu, C., et. al., Nature Biotech.25(2007) 1290-1297).

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

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

In one approach, bispecific antibodies that closely resemble native antibodies have been generated using a four-source hybridoma technology based on somatic fusion of two different hybridoma cell lines that express a murine monoclonal antibody with the desired specificity for the bispecific antibody (see Milstein, c., and Cuello, a.c., Nature 305(1983) 537-540). Due to the random pairing of two different antibody weights and light chains within the resulting hybridoma-hybridoma (or quadroma) cell lines, up to 10 different antibody species were generated, only one of which was the desired functional bispecific antibody. Due to the presence of mis-paired byproducts and significantly reduced production yields, elaborate purification procedures are required (see, e.g., Morrison, S.L., Nature Biotech.25(2007) 1233-1234). In general, the same problem of mis-pairing of by-products remains if recombinant expression techniques are used.

One way to circumvent the problem of mis-pairing by-products, known as 'knob-into-hole', aims at forcing the pairing of two different antibody heavy chains by introducing mutations into the CH3 domain to modify the contact interface. On one strand, large volumes of amino acids are replaced with amino acids with short side chains to create 'holes'. Instead, an amino acid with a large side chain was introduced into another CH3 domain to create a 'knot'. By co-expressing both heavy chains (and both identical light chains, which must be adapted to both heavy chains), high yields of heterodimer formation ('node-hole') versus homodimer formation ('hole-hole' or 'node-node') were observed (Ridgway, J.B., et al., Protein Eng.9(1996) 617-621; and WO 96/027011). The percentage of heterodimers can be further increased by reshaping the interaction surface of the two CH3 domains and introducing disulfide bridges to stabilize the heterodimers using a phage display approach (Merchant, A.M., et al., Nature Biotech.16(1998) 677-681; Atwell, S., et al., J.mol.biol.270(1997) 26-35). For example, EP 1870459A 1 describes a new method of the knot-in-point technique. While this format appears very attractive, no data describing progress toward the clinic is currently available. An important constraint of this strategy is that the light chains of both parent antibodies have to be identical to prevent mispairing and inactive molecule formation. Thus, this technique is not suitable as a basis for the easy development of recombinant tri-or tetra-specific antibodies against three or four antigens starting from two antibodies against a first and a second antigen, since the heavy chains and/or the same light chains of these antibodies have to be optimized first, and then further antigen-binding peptides against a third and a fourth antigen have to be added.

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

Summary of The Invention

The present invention relates to a trispecific or tetraspecific antibody comprising:

a) a light chain and a heavy chain of a full-length antibody that specifically binds a first antigen; and

b) a modified light chain and a modified heavy chain of a full-length antibody that specifically binds a second antigen, wherein the variable domains VL and VH are replaced with each other, and/or wherein the constant domains CL and CH1 are replaced with each other; and

c) wherein one to four antigen binding peptides that specifically bind to one or two additional antigens are fused via a peptide linker to the C or N terminus of the light or heavy chain of a) and/or b).

Another embodiment of the invention is a method for preparing a trispecific or tetraspecific antibody according to the invention, comprising the steps of:

a) transformation of host cells with vectors comprising nucleic acid molecules encoding

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

ab) a modified light chain and a modified heavy chain of a full-length antibody that specifically binds a second antigen, wherein the variable domains VL and VH are replaced with each other, and/or wherein the constant domains CL and CH1 are replaced with each other; and

ac) wherein one to four antigen binding peptides that specifically bind to one or two additional antigens are fused via a peptide linker to the C or N terminus of the light or heavy chain of a) and/or b);

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

c) Recovering the antibody molecule from the culture.

Another embodiment of the invention is a host cell comprising a vector comprising a nucleic acid molecule encoding:

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

c) one to four antigen binding peptides that specifically bind to the other one or two antigens are fused via a peptide linker to the C or N terminus of the light or heavy chain of a) and/or b).

Another embodiment of the invention is a composition, preferably a pharmaceutical composition or a 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 the patient.

In accordance with the present invention, the ratio of desired tri-or tetra-specific antibodies to undesired by-products can be improved by replacing certain domains only in the heavy and light chain pairs (HC/LC) of a full length antibody that specifically binds a second antigen (second antibody). In this way, unwanted incorrect pairing of light chains with incorrect heavy chains (light chain of the first antibody with heavy chain of the second antibody or light chain of the second antibody with heavy chain of the first antibody) can be reduced.

Detailed Description

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention comprises one or two antigen binding peptides specifically binding to one or two further antigens under c).

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention is characterized in that said antigen binding peptide is selected from the group consisting of: scFv fragments and scFab fragments.

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention is characterized in that the antigen binding peptide is a scFv fragment.

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention is characterized in that said antigen binding peptide is a scFab fragment.

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention is characterized in that said antigen binding peptide is fused to the C-terminus of the heavy chain of a) and/or b).

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention comprises under c) one or two antigen binding peptides specifically binding one further antigen.

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention comprises under c) two identical antigen binding peptides that specifically bind to a third antigen. Preferably, the two identical antigen binding peptides are both fused to the C-terminus of the heavy chains of a) and b) via the same peptide linker. Preferably, the two identical antigen binding peptides are scFv fragments or scFab fragments.

In one embodiment of the invention, the trispecific or tetraspecific antibody according to the invention comprises under c) two antigen binding peptides specifically binding to a third and a fourth antigen. In one embodiment, the two antigen binding peptides are both fused to the C-terminus of the heavy chains of a) and b) via the same peptide linker. Preferably, the two antigen binding peptides are scFv fragments or scFab fragments.

In accordance with the present invention, the ratio of a desired tri-or tetra-specific antibody to an undesired by-product (due to mispairing of the light chain with the "wrong" heavy chain of an antibody that specifically binds another antigen) can be improved by replacing only certain domains in a pair of heavy and light chains (HC/LC). While the first pair of the two full-length HC/LC pairs is derived from an antibody that specifically binds to a first antigen and remains substantially unchanged, the second pair of the two full-length HC/LC pairs is derived from an antibody that specifically binds to a second antigen and is modified by the following substitutions:

-light chain: the variable light chain domain VL is replaced with the variable heavy chain domain VH of said antibody which specifically binds to the second antigen, and/or the constant light chain domain CL is replaced with the constant heavy chain domain CH1 of said antibody which specifically binds to the second antigen, and

-heavy chain: the variable heavy domain VH is replaced with the variable light domain VL of said antibody that specifically binds to the second antigen, and/or the constant heavy domain CH1 is replaced with the constant light domain CL of said antibody that specifically binds to the second antigen.

Bispecific antibodies with improved ratios for this are then fused via peptide linkers to the C or N terminus of the light or heavy chains of the two antibodies that specifically bind the first and second antigens with one to four antigen-binding peptides that specifically bind the other one or two antigens, resulting in trispecific and tetraspecific antibodies according to the invention.

Thus, the resulting trispecific and tetraspecific antibodies according to the invention are artificial antibodies comprising:

b) a light chain and a heavy chain of an antibody that specifically binds a second antigen, wherein the light chain (of the antibody that specifically binds the second antigen) contains the variable domain VH instead of VL and/or the constant domain CH1 instead of CL, and wherein the heavy chain (of the antibody that specifically binds the second antigen) contains the variable domain VL instead of VH and/or the constant domain CL instead of CH 1.

In another aspect of the invention, the improved ratio of the desired bivalent bispecific antibody compared to the undesired side products can be further improved by modifying the CH3 domain of the full length antibody specifically binding the first and second antigens in the three or four specific antibodies.

Thus, in a preferred embodiment of the invention, the CH3 domain of the tri-or tetraspecific antibodies according to the invention (heavy chain neutralized and modified heavy chain) may be altered by the "knob-into-knob" technique, which is described in detail in, for example, WO 96/027011, Ridgway, J.B., et al, Protein Eng.9(1996) 617-621; and Merchant, a.m., et al, nat. biotechnol.16(1998) 677-. In this approach, the interaction interface of the two CH3 domains is altered to enhance heterodimerization of the two heavy chains containing the two CH3 domains. Each of the two CH3 domains (of both heavy chains) may be a "knot" and the other a "hole". Introduction of disulfide bridges further stabilized the heterodimer (Merchant, A.M., et al., Nature Biotech.16(1998) 677-.

Thus, in one aspect of the invention, the trispecific or tetraspecific antibody is further characterized by

a) The CH3 domain of the heavy chain of the full-length antibody of b) and the CH3 domain of the modified heavy chain of the full-length antibody of b) meet at an interface comprising the initial interface between the CH3 domains of the antibody; wherein the interface is altered to facilitate formation of a trispecific or tetraspecific antibody, wherein the alteration is characterized by:

i) altering the CH3 domain of one heavy chain such that within the initial interface of the CH3 domain of the other heavy chain encountered in the three or four specific antibody within the CH3 domain of one heavy chain, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the interface of the CH3 domain of one heavy chain that can locate in a cavity within the interface of the CH3 domain of the other heavy chain;

and is

ii) altering the CH3 domain of the other heavy chain such that within the initial interface of the first CH3 domain in the second CH3 domain encountered in the three or four specific antibody, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain wherein the protuberance 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 with 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 by introducing cysteine (C) as an amino acid in the corresponding position of each CH3 domain, enabling the formation of a disulfide bridge between the two CH3 domains.

In a preferred embodiment, the trispecific or tetraspecific antibody comprises a T366W mutation in the CH3 domain of "knot chain" and a T366S, L368A, Y407V mutation in the CH3 domain of "pocket chain". Another interchain disulfide bridge between CH3 domains (Merchant, a.m., et al, nature biotech.16(1998) 677-. Thus, in another preferred embodiment, said trispecific or tetraspecific antibody comprises the Y349C, T366W mutation in one of said two CH3 domains and the E356C, T366S, L368A, Y407V mutation in the other of said two CH3 domains, or said trispecific or tetraspecific antibody comprises the Y349C, T366W mutation in one of the two CH3 domains and the S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains (the other Y349C mutation in one CH3 domain and the other E356C or S354C mutation in the other CH3 domain form inter-chain disulfide bridges) (numbering always according to the index EU of Kabat). However, other node-in-hole techniques, such as those described in EP 1870459 a1, may alternatively or additionally be used. A preferred example of such a trispecific or tetraspecific antibody is R409D in the "chain-binding" CH3 domain; the K370E mutation and D399K in the "pocket chain" CH3 domain; the E357K mutation (numbering is always according to EU index of Kabat).

In another preferred embodiment, the trispecific or tetraspecific antibody comprises a T366W mutation in the "chain-forming" CH3 domain and a T366S, L368A, Y407V mutation in the "pocket chain" CH3 domain and additionally R409D in the "chain-forming" CH3 domain; the K370E mutation and D399K in the "pocket chain" CH3 domain; the E357K mutation.

In another preferred embodiment, the trispecific or tetraspecific antibody comprises a Y349C, a T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains, or the trispecific or tetraspecific antibody comprises a Y349C, a T366W mutation in one of the two CH3 domains and a S354C, T366S, L368A, Y407V mutation in the other of the two CH3 domains and additionally a R409D in the "chain binding" CH3 domain; the K370E mutation and D399K in the "pocket chain" CH3 domain; the E357K mutation.

The term "full-length antibody" refers to an antibody consisting of two antibody heavy chains and two antibody light chains (see fig. 1). The heavy chain of the full-length antibody is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody heavy chain constant domain 1(CH1), an antibody Hinge Region (HR), an antibody heavy chain constant domain 2(CH2), and an antibody heavy chain constant domain 3(CH3) in the N-terminal to C-terminal direction, abbreviated as VH-CH1-HR-CH2-CH 3; in the case of antibodies of subclass IgE, there is also an optional antibody heavy chain constant domain 4(CH 4). Preferably, the heavy chain of the full length antibody is a polypeptide consisting of VH, CH1, HR, CH2 and CH3 in the N-terminal to C-terminal direction. The light chain of a full-length antibody is a polypeptide consisting of an antibody light chain variable domain (VL) and an antibody light chain constant domain (CL), abbreviated VL-CL, in the N-terminal to C-terminal direction. The antibody light chain constant domain (CL) may be kappa (kappa) or lambda (lambda). Full-length antibody chains are linked together via interpoly disulfide bonds between the CL domain and the CH1 domain (i.e., between the light and heavy chains) and between the full-length antibody heavy chain hinge region. Examples of typical full-length antibodies are natural antibodies like IgG (e.g., IgG1 and IgG2)IgM, IgA, IgD, and IgE. Full length antibodies according to the invention may be from a single species, e.g., human, or they may be chimeric or humanized antibodies. A full-length antibody according to the invention comprises two antigen-binding sites, each formed by a VH and VL pair, both of which specifically bind the same antigen. The C-terminus of the heavy or light chain of the full-length antibody represents the last amino acid at the C-terminus of the heavy or light chain. As used within the present invention, the term "peptide linker" refers to a peptide having an amino acid sequence of preferred synthetic origin. These peptide linkers according to the invention are used to fuse antigen binding peptides to the C-or N-terminus of full-length and/or modified full-length antibody chains to form trispecific or tetraspecific antibodies according to the invention. Preferably, the peptide linker under c) is a peptide having an amino acid sequence of at least 5 amino acids in length, preferably 5 to 100, more preferably 10 to 50 amino acids in length. In one embodiment, the peptide linker is (G)_xS)_nOr (G)_xS)_nG_mWhere G ═ glycine, S ═ serine, and (x ═ 3, n ═ 3, 4, 5, or 6, and m ═ 0, 1, 2, or 3) or (x ═ 4, n ═ 2, 3, 4, or 5, and m ═ 0, 1, 2, or 3), preferably, x ═ 4 and n ═ 2 or 3, more preferably, x ═ 4, n ═ 2. In one embodiment, the peptide linker is (G)₄S)₂。

As used, the term "antigen-binding peptide" refers to a monovalent antigen-binding fragment or derivative of a full-length antibody, which includes an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), or a VH/VL pair derived from a full-length antibody, or an antibody fragment such as a VH domain and/or a VL domain, a single chain fv (scfv) fragment, or a single chain fab (scfab) fragment. Preferably, the antigen binding peptide comprises at least an antibody heavy chain variable domain (VH) and an antibody light chain variable domain (VL). In a preferred embodiment, such antigen binding peptides are selected from the group consisting of: VH domains, single chain fv (scfv) fragments, and single chain fab (scfab) fragments, preferably selected from the group consisting of: single chain fv (scfv) fragments and single chain fab (scfab) fragments.

As used herein, the term "binding site" or "antigen binding site" refers to the region of an antibody molecule to which a ligand (e.g., an antigen or antigenic fragment thereof) actually binds and which is derived from the antibody. The antigen binding site comprises an antibody heavy chain variable domain (VH) and/or an antibody light chain variable domain (VL), or a VH/VL pair.

The antigen binding site that specifically binds to the desired antigen may be derived from a) known antibodies against the antigen or b) novel antibodies or antibody fragments obtained by the de novo immunization method using antigen proteins or nucleic acids or fragments thereof or the like or by phage display.

The antigen binding site of an antibody of the invention may contain six Complementarity Determining Regions (CDRs) that contribute to varying degrees to the affinity of the binding site for the antigen. There are three heavy chain variable domain CDRs (CDRH1, CDRH2 and CDRH3) and three light chain variable domain CDRs (CDRL1, CDRL2 and CDRL 3). The extent of the CDR and Framework Regions (FR) is determined by comparison with a compiled database of amino acid sequences in which those regions have been defined in terms of variability between sequences. Also included within the scope of the invention are functional antigen binding sites that are made up of fewer CDRs (i.e., where binding specificity is determined by three, four, or five CDRs). For example, less than the full set of 6 CDRs may be sufficient for binding. In some cases, a VH or VL domain may be sufficient.

Antibody specificity refers to the selective recognition of a particular epitope by an antibody. For example, natural antibodies are monospecific. Bispecific antibodies are antibodies with two different antigen binding specificities. Trispecific antibodies are thus antibodies of the invention with three different antigen binding specificities. Tetraspecific antibodies according to the invention are antibodies with four different antigen binding specificities.

If the antibody has more than one specificity, the recognized epitope may be associated with one antigen or with more than one antigen.

As used herein, the term "monospecific" antibody refers to an antibody having one or more binding sites, each of which binds to the same epitope of the same antigen.

As used within this application, the term "valency" or "valency" means the presence of the specified number of binding sites in an antibody molecule. For example, a natural antibody or a full-length antibody according to the invention has two binding sites and is bivalent. Thus, the term "trivalent" means that there are three binding sites in an antibody molecule. As used herein, the term "trivalent trispecific" antibody refers to an antibody having three antigen binding sites, each binding to another antigen (or another epitope of the antigen). The antibodies of the invention have three to six binding sites, i.e., are tri-, tetra-, penta-, or hexavalent (preferably tri-or tetravalent) and are tri-or tetraspecific.

An "scFv fragment" or "single chain Fv fragment" (see figure 2b) is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody light chain variable domain (VL), and a single chain Fv linker, wherein the antibody domain and the single chain Fv linker have one of the following sequences in the N-terminal to C-terminal direction: a) a VH-single chain Fv linker-VL, b) a VL-single chain Fv linker-VH; preferably a) a VH-single chain Fv linker-VL, and wherein said single chain Fv linker is a polypeptide having an amino acid sequence of at least 15 amino acids in length, in one embodiment at least 20 amino acids in length. The term "N-terminal" denotes the last amino acid from the N-terminus and the term "C-terminal" denotes the last amino acid from the C-terminus.

As used within single-chain Fv fragments, the term "single-chain Fv linker" denotes a peptide having an amino acid sequence of preferred synthetic origin. The single chain Fv linker is a peptide having an amino acid sequence of at least 15 amino acids in length, in one embodiment at least 20 amino acids in length, and preferably between 15 and 30 amino acids in length. In one embodiment, the single-link joint is (G)_xS)_nWhere G ═ glycine, S ═ serine, (x ═ 3 and n ═ 4, 5, or 6) or (x ═ 4 and n ═ 3, 4, 5, or 6), preferably, x ═ 4, n ═ 3, 4, or 5, more preferably, x ═ 4, n ═ 3, or 4. In one embodiment, the single chain Fv linker is (G)₄S)₃Or (G)₄S)₄。

In addition, the single chain Fv fragment is preferably disulfide-stabilized. Such further disulfide stabilization of single chain antibodies is achieved by introducing disulfide bonds between the variable domains of single chain antibodies and is described, for example, in WO 94/029350, Rajagopal, V.et al, prot. Engin.10(1997) 1453-; kobayashi, H., et al, nucleic Medicine & Biology 25(1998) 387-393; or Schmidt, M., et al, Oncogene 18(1999) 1711-.

In one embodiment of the disulfide-stabilized single chain Fv fragment, the disulfide bond between the variable domains of the single chain Fv fragment comprised in the antibody according to the invention is, for each single chain Fv fragment, independently selected from the group consisting of:

i) heavy chain variable domain position 44 to light chain variable domain position 100,

ii) heavy chain variable domain position 105 to light chain variable domain position 43, or

iii) heavy chain variable domain position 101 to light chain variable domain position 100.

In one embodiment, the disulfide bond between the variable domains of a single chain Fv fragment comprised in an antibody according to the invention is between heavy chain variable domain position 44 and light chain variable domain position 100.

A "scFab fragment" or "single chain Fab fragment" (see fig. 2a) is a polypeptide consisting of an antibody heavy chain variable domain (VH), an antibody constant domain 1(CH1), an antibody light chain variable domain (VL), an antibody light chain constant domain (CL) and a linker, wherein the antibody domain and the linker have one of the following sequences in the N-terminal to C-terminal direction: a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1 or d) VL-CH 1-linker-VH-CL; and wherein the linker is a polypeptide of at least 30 amino acids, preferably between 32 and 50 amino acids. The single chain Fab fragment a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1 and d) VL-CH 1-linker-VH-CL is stabilized by the natural disulfide bond between the CL domain and the CH1 domain. The term "N-terminal" denotes the last amino acid from the N-terminus and the term "C-terminal" denotes the last amino acid from the C-terminus.

As used within the present invention, the term "linker" denotes an amino acid sequence having a preferred synthetic originThe peptide of (1). These peptides according to the invention are used to link a) VH-CH1 to VL-CL, b) VL-CL to VH-CH1, c) VH-CL to VL-CH1 or d) VL-CH1 to VH-CL to form the following single-chain Fab fragments according to the invention: a) VH-CH 1-linker-VL-CL, b) VL-CL-linker-VH-CH 1, c) VH-CL-linker-VL-CH 1 or d) VL-CH 1-linker-VH-CL. The linker within the single chain Fab fragment is a peptide having an amino acid sequence of at least 30 amino acids in length, preferably 32 to 50 amino acids in length. In one embodiment, the linker is (G)_xS)_nWhere G ═ glycine, S ═ serine, (x ═ 3, n ═ 8, 9, or 10 and m ═ 0, 1, 2, or 3) or (x ═ 4 and n ═ 6, 7, or 8 and m ═ 0, 1, 2, or 3), preferably, x ═ 4, n ═ 6, or 7 and m ═ 0, 1, 2, or 3, more preferably, x ═ 4, n ═ 7 and m ═ 2. In one embodiment, the linker is (G)₄S)₆G₂。

In a preferred embodiment, the antibody domain and the linker in the single chain Fab fragment have one of the following orders in the N-terminal to C-terminal direction: a) VH-CH 1-linker-VL-CL, or b) VL-CL-linker-VH-CH 1, more preferably, VL-CL-linker-VH-CH 1.

In another preferred embodiment, said antibody domain and said linker in said single chain Fab fragment have one of the following orders in the N-terminal to C-terminal direction: a) VH-CL-linker-VL-CH 1 or b) VL-CH 1-linker-VH-CL.

Optionally, in the single chain Fab fragment, in addition to the natural disulfide bond between the CL domain and the CH1 domain, the antibody heavy chain variable domain (VH) and the antibody light chain variable domain (VL) are disulfide-stabilized by introducing a disulfide bond between the following positions:

iii) heavy chain variable domain position 101 to light chain variable domain position 100 (numbering is always according to the EU index of Kabat).

Such further disulphide stabilization of single chain Fab fragments is achieved by introducing a disulphide bond between the variable domains VH and VL of the single chain Fab fragment. Techniques for introducing non-natural disulfide bridges for the stabilization of single-chain Fv's are described, for example, in WO 94/029350, Rajagopal et al, prot. Engin.10(1997) 1453-; kobayashi et al, nucleic Medicine & Biology 25(1998) 387-393; or Schmidt et al, Oncogene 18(1999) 1711-1721. In one embodiment, the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the present invention is between heavy chain variable domain position 44 and light chain variable domain position 100. In one embodiment, the optional disulfide bond between the variable domains of the single chain Fab fragments comprised in the antibody according to the present invention is between heavy chain variable domain position 105 and light chain variable domain position 43 (numbering is always according to the EU index of Kabat).

In one embodiment, single chain Fab fragments are preferred without said optional disulfide stabilized between the variable domains VH and VL of the single chain Fab fragment.

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

As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules of a single amino acid composition.

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

The term "humanized antibody" refers to an antibody in which the framework or "complementarity determining regions" (CDRs) are modified to comprise the CDRs of an immunoglobulin of different specificity compared to the specificity of the parent immunoglobulin. In a preferred embodiment, murine CDRs are grafted into the framework regions of a human antibody to make a "humanized antibody". See, e.g., Riechmann, L., et al., Nature 332(1988) 323-327; and Neuberger, M.S., et al., Nature 314(1985) 268-270. Other forms of "humanized antibodies" encompassed by the present invention are those in which the constant regions have been additionally modified or altered relative to the constant regions of the original antibody to generate properties in accordance with 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., curr. opin. chem. biol.5(2001) 368-374). Human antibodies can also be produced in transgenic animals (e.g., mice) that are capable of producing a complete repertoire or selection of human antibodies in the absence of endogenous immunoglobulin production upon immunization. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice results in the production of human antibodies upon antigen challenge (see, e.g., Jakobovits, A., et al., Proc. Natl. Acad. Sci. USA 90(1993) 2551-. Human antibodies can also be generated in phage display libraries (Hoogenboom, H.R., and Winter, G., J.Mol.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 (1985); and Boerner, P.et al, J.Immunol.147(1991) 86-95). As already mentioned in connection with the chimeric and humanized antibodies according to the invention, the term "human antibody" as used herein also includes such antibodies which have been modified in the constant region to generate the properties according to the invention, in particular with respect to C1q binding and/or FcR binding, for example by "class switching", i.e. by alteration or mutation of the Fc part (e.g. from IgG1 to IgG4 and/or IgG1/IgG4 mutations).

As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created or isolated by recombinant means, as is an antibody isolated from a host cell such as NS0 or CHO cells or from an animal (e.g., mouse) transgenic for human immunoglobulin genes or expressed using a recombinant expression vector transfected into a host. Such recombinant human antibodies have rearranged forms of variable and constant regions. Recombinant human antibodies according to the invention have been subjected to 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 a human antibody germline repertoire.

As used herein, "variable domain" (variable domain of light chain (VL), variable domain of heavy chain (VH)) means each of the light and heavy chain pairs that are directly involved in binding of an antibody to an antigen. The variable domains of human light and heavy chains have the same general structure, and each domain comprises four Framework (FR) regions whose sequences are widely conserved, connected by three "hypervariable regions" (or complementarity determining regions, CDRs). The framework regions adopt a β -sheet conformation, while the CDRs may form loops, connecting the β -sheet structure. 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 therefore provide another object of the invention.

As used herein, the term "hypervariable region" or "antigen-binding portion of an antibody" refers to the amino acid residues of an antibody which are responsible for antigen-binding. Hypervariable regions comprise amino acid residues from "complementarity determining regions" or "CDRs". The "framework" or "FR" regions are those regions of the variable domain other than the hypervariable region residues defined herein. Thus, the light and heavy chain N-to-C termini of antibodies comprise domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR 4. The CDRs on each chain are separated by such framework amino acids. In particular, CDR3 of the heavy chain is the region that contributes most to antigen binding. CDR and FR regions were determined according to the standard definition of Kabat, et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

As used herein, the term "binding"/"specific binding" refers to the binding of an antibody to an epitope in an in vitro assay, preferably a plasmon resonance assay (BIAcore, GE-healthcare uppsala, Sweden) using a purified wild-type antigen. Binding affinity is given by the term k_a(rate constant of binding of antibody from antibody/antigen Complex), k_D(dissociation constant), and K_D(k_D/k_a) To be defined. In one embodiment, binding or specific binding means 10^-8mol/l or less, preferably 10^-9M to 10^-13Binding affinity (K) in mol/l_D). Thus, a tri-or tetra-specific antibody according to the invention is preferably present at 10^-8mol/l or less, preferably 10^-9To 10^-13Binding affinity (K) in mol/l_D) Specifically binding to each of the antigens to which it is specific.

Binding of antibodies to Fc γ RIII can be investigated by BIAcore assay (GE-Healthcare Uppsala, Sweden). Binding affinity is given by the term k_a(rate constant of binding of antibody from antibody/antigen Complex), k_D(dissociation constant), and K_D(k_D/k_a) To be defined.

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

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

In another embodiment, the tri-or tetra-specific antibody according to the invention is characterized in that the full-length antibody is of the subclass human IgG1 or human IgG1 with the mutations L234A and L235A.

In another embodiment, a tri-or tetra-specific antibody according to the invention is characterized in that the full-length antibody is of the subclass human IgG 2.

In another embodiment, a tri-or tetra-specific antibody according to the invention is characterized in that the full-length antibody is of the subclass human IgG 3.

In another embodiment, the tri-or tetra-specific antibody according to the invention is characterized in that the full-length antibody is of the subclass human IgG4 or of the subclass human IgG4 with the further mutation S228P.

Preferably, the tri-or tetra-specific antibody according to the invention is characterized in that the full-length antibody is of the subclass human IgG1 or of the subclass human IgG4 with the further mutation S228P.

It has now been found that tri-or tetra-specific antibodies according to the invention have improved characteristics, such as biological or pharmacological activity, pharmaceutical properties or toxicity. They are useful, for example, in the treatment of diseases, such as cancer.

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

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

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

In one embodiment, the antibody according to the invention has reduced FcR binding compared to the IgG1 antibody. Thus, with respect to FcR binding, the full-length parent antibody is of the IgG4 subclass or of the IgG1 or IgG2 subclass with mutations in S228, L234, L235 and/or D265, and/or contains a PVA236 mutation. In one embodiment, the mutation in the full-length parent antibody is S228P, L234A, L235A, L235E, and/or PVA 236. In another embodiment, the mutations in the full-length parent antibody are in IgG4S228P and in IgG1L234A and L235A.

The constant regions of antibodies are directly involved in ADCC (antibody-dependent cell-mediated cytotoxicity) and CDC (complement-dependent cytotoxicity). Complement activation (CDC) is initiated by the binding of complement factor C1q to the constant regions of most IgG antibody subclasses. C1q binding to antibodies is caused by defined protein-protein interactions at the so-called binding site. Such constant region binding sites are known in the art and are described, for example, in Lukas, T.J., et al, J.Immunol.127(1981) 2555-2560; bunkhouse, r.and Cobra, j.j., mol.immunol.16(1979) 907-; burton, D.R., et al, Nature 288(1980) 338-344; thomson, j.e., et al, mol. immunol.37(2000) 995-; idiocies, e.e., et al, j.immunol.164(2000) 4178-; hearer, M., et al, J.Virol.75(2001) 12161-; morgan, A., et al, Immunology86(1995)319- "324; and EP 0307434. Such constant region binding sites are characterized, for example, by amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to the EU index of Kabat).

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

The term "Complement Dependent Cytotoxicity (CDC)" refers to a process initiated by the binding of complement factor C1q to the Fc portion of most IgG antibody subclasses. C1q binding to antibodies is caused by defined protein-protein interactions at the so-called binding site. Such Fc moiety binding sites are known in the art (see above). Such Fc moiety binding sites are characterized by, for example, amino acids L234, L235, D270, N297, E318, K320, K322, P331, and P329 (numbering according to EU index of Kabat). Antibodies of subclasses IgG1, IgG2, and IgG3 typically show complement activation, including C1q and C3 binding, while IgG4 does not activate the complement system and does not bind C1q and/or C3.

The cell-mediated effector functions of monoclonal antibodies can be enhanced by engineering their oligosaccharide components as described by Umana, P., et al, Nature Biotechnol.17(1999)176-180 and U.S. Pat. No. 6,602,684. IgG 1-type antibodies, the most commonly used therapeutic antibodies, are glycoproteins with conserved N-linked glycosylation sites at Asn297 of each CH2 domain. Two complex biantennary oligosaccharides attached to Asn297 are buried between the CH2 domains, making extensive contact with the polypeptide backbone, and their presence is crucial for antibody-mediated effector functions such as antibody-dependent cellular cytotoxicity (ADCC) (Lifely, m., r., et al, Glycobiology 5(1995) 813-822; Jefferis, r., et al, immunol. rev.163(1998) 59-76; Wright, a., and Morrison, s.l., Trends biotechnol.15(1997) 26-32). Umana, p, et al, Nature biotechnol.17(1999)176-180 and WO 99/54342 show that overexpression of β (1, 4) -N-acetylglucosaminyltransferase III ("GnTIII"), a glycosyltransferase that catalyzes the formation of bisected oligosaccharides, significantly increases the in vitro ADCC activity of antibodies in Chinese Hamster Ovary (CHO) cells. Changes in the composition of Asn297 carbohydrates or elimination thereof also affect binding to Fc γ R and C1q (Umana, P., et al., Nature Biotechnol.17(1999) 176-.

Methods for enhancing cell-mediated effector function of monoclonal antibodies are reported in, for example, WO2005/018572, WO 2006/116260, WO 2006/114700, WO 2004/065540, WO2005/011735, WO 2005/027966, WO 1997/028267, US 2006/0134709, US2005/0054048, US 2005/0152894, WO 2003/035835, WO 2000/061739.

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

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

As used within this application, the term "NGNA" denotes the sugar residue N-glycolyl neuraminic acid.

Glycosylation of human IgG1 or IgG3 occurred at Asn297, with the core fucosylated biantennary complex oligosaccharide being glycosylated with up to two Gal residues as termini. Human constant heavy chain regions of the IgG1 or IgG3 subclasses are reported in detail in Kabat, E.E., A., et al, Sequences of Proteins of Immunological Interest, 5th Ed. public Health Service, National Institutes of Health, Bethesda, MD. (1991) and Bruggemann, M.et al, J.Exp.Med.166 (1981367) 1351-; love, T., W., et al, Methods enzymol.178(1989) 515-. Depending on the amount of terminal Gal residues, these structures are referred to as G0, G1 (. alpha. -1, 6-or. alpha. -1, 3-), or G2 glycan residues (Raju, T., S., Bioprocess int.1(2003) 44-53). CHO-type glycosylation of the Fc portion of antibodies is described, for example, in Router, F.H., Glycoconjugate J.14(1997) 201-207. Antibodies recombinantly expressed in CHO host cells that are not sugar modified are typically fucosylated at Asn297 in an amount of at least 85%. The modified oligosaccharides of the full-length parent antibody may be hybrid or complex. Preferably, the bisected reduced/nonfucosylated oligosaccharides are hybrid. In another embodiment, the bisected reduced/nonfucosylated oligosaccharides are complex.

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

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

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

Amino acid sequence variants (or mutants) of tri-or tetra-specific antibodies are prepared by introducing appropriate nucleotide changes into antibody DNA or by nucleotide synthesis. Such modifications may be carried out, however, only to a very limited extent, for example as described above. For example, the modifications do not alter the antibody characteristics described above, such as IgG isotype and antigen binding, but may improve yield of recombinant production, protein stability, or facilitate purification.

As used herein, the term "host cell" refers to any class of cellular system that can be engineered to produce antibodies according to the present invention. In one embodiment, HEK293 cells and CHO cells are used as host cells. As used herein, the expressions "cell," "cell line," and "cell culture" are used interchangeably, and all such references include progeny. Thus, the words "transformant" and "transformed cell" include the primary test cell and cultures derived therefrom, regardless of the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for the originally transformed cell are included. Where different designations are intended, it will be clear from the context.

Expression in NS0 cells is described, for example, in Barnes, l.m., et al, Cytotechnology 32(2000) 109-123; barnes, L.M., et al, Biotech.Bioeng.73(2001) 261-. Transient expression is described, for example, in Durocher, y., et al, nucleic. Cloning of the variable domains is described in Orlandi, r., et al, proc.natl.acad.sci.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 204(1997) 77-87. A preferred transient expression system (HEK 293) is described in Schlaeger, E.J., and Christensen, K., in Cytotechnology 30(1999)71-83 and Schlaeger, E.J., J.Immunol. methods 194(1996) 191-199.

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

A nucleic acid is "operably linked" when it is in a functional relationship with another nucleic acid sequence. For example, if a DNA of a pre-sequence (pre-sequence) or a secretion leader (secretory leader) is expressed as a pre-protein (pre-protein) involved in the secretion of the polypeptide, it is operably linked to the DNA of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of that sequence; alternatively, if the ribosome binding site is positioned to facilitate translation, it is operably linked to a coding sequence. Generally, "operably linked" means that the DNA sequences being linked are contiguous and, in the case of a secretory leader, contiguous and in reading order. However, enhancers need not be contiguous. Ligation may be achieved by ligation reactions at convenient restriction sites. If there are no such sites, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

Purification of the antibody to remove cellular components or other contaminants, such as other cellular nucleic acids or proteins, is performed by standard techniques, including alkali/SDS treatment, CsCl banding, column chromatography, agarose gel electrophoresis, and other techniques well known in the art. See, Ausubel, F., et al, ed. Current Protocols in molecular Biology, Greene Publishing and Wiley Interscience, New York (1987). Different methods are well established and widely used for protein purification, such as affinity chromatography using microbial proteins (e.g. protein a or protein G affinity chromatography), ion exchange chromatography (e.g. cation exchange (carboxymethyl resin), anion exchange (aminoethyl resin) and mixed mode exchange), thiophilic adsorption (e.g. using beta mercaptoethanol and other SH ligands), hydrophobic interaction or aromatic adsorption chromatography (e.g. using phenyl-sepharose, aza-arsenophilic resin, or metanilic acid), metal chelate affinity chromatography (e.g. using ni (ii) -and cu (ii) -affinity materials), size exclusion chromatography, and electrophoretic methods (such as gel electrophoresis, capillary electrophoresis) (vijayaakshmi, m.a., appl. biochem. biotech.75(1998) 93-102).

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

One embodiment of the invention is a tri-or tetra-specific antibody according to the invention for use in the treatment of cancer.

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

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

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

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

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

As used herein, the phrase "parenteral administration" means modes of administration other than enteral and topical administration, typically by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural, and intrasternal injection and infusion.

As used herein, the term cancer refers to a proliferative disease, such as lymphoma, lymphocytic leukemia, lung cancer, non-small cell lung (NSCL) cancer, bronchoalveolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, head and neck cancer, cutaneous or intraocular melanoma, uterine cancer, ovarian cancer, rectal cancer, cancer of the anal region, stomach cancer, cancer of the stomach, colon cancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, hodgkin's disease, carcinoma of the esophagus, carcinoma of the small intestine, cancer of the endocrine system, carcinoma of the thyroid gland, carcinoma of the parathyroid gland, carcinoma of the adrenal gland, sarcoma of soft tissue, carcinoma of the urethra, carcinoma of the penis, prostate cancer, carcinoma of the bladder, carcinoma of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular carcinoma, carcinoma of the gallbladder, Central Nervous System (CNS) neoplasms, spinal column tumors, brain stem glioma, glioblastoma multif, Astrocytoma, schwannoma, ependymoma, medulloblastoma, meningioma, squamous cell carcinoma, pituitary adenoma, and ewing's sarcoma, including refractory forms of any of the foregoing cancers, or a combination of one or more of the foregoing cancers.

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

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

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

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

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

As used herein, the term "transformation" refers to the process of vector/nucleic acid transfer into a host cell. If cells without intractable cell wall barriers are used as host cells, they are transfected, for example, by calcium phosphate precipitation, as described by Graham and Van der Eh, Virology 52(1978)546 below. However, other methods for introducing DNA into cells may also be used, such as by nuclear injection or by protoplast fusion. If prokaryotic cells or cells containing a rigid cell wall structure are used, one method of transfection is, for example, calcium treatment with calcium chloride, as described in Cohen, F.N, et al, PNAS 69(1972)7110, and the like.

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

A "vector" is a nucleic acid molecule, particularly self-replicating, that transfers an inserted nucleic acid molecule into and/or between host cells. The term includes vectors that function primarily to insert DNA or RNA into a cell (e.g., chromosomal integration), primarily 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 providing more than one of said functions.

An "expression vector" is a polynucleotide capable of transcription and translation into a polypeptide when introduced into an appropriate host cell. An "expression system" generally refers to a suitable host cell comprising an expression vector that is capable of functioning to produce a desired expression product.

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

Description of the amino acid sequence

SEQ ID NO: 1 light chain < Ang-2>

SEQ ID NO: 2 knob-heavy chain < Ang-2 of < EGFR > scFv with C-terminal fusion

SEQ ID NO: 3 light chain with CH1-CL crossover < VEGF >

SEQ ID NO: hole-heavy chain of < IGF-1R > scFv with CH1-CL exchange and C-terminal fusion

<VEGF>

SEQ ID NO: 5 knot-heavy chain of < EGFR > scFab with C-terminal fusion < Ang-2>

SEQ ID NO: 6 pocket-heavy chain of < IGF-1R > scFab with CH1-CL exchange and C-terminal fusion

<VEGF>

SEQ ID NO: 7 pocket-heavy chain of < EGFR > scFv with CH1-CL exchange and C-terminal fusion

<VEGF>

SEQ ID NO: 8 hole-heavy chain with CH1-CL exchange < VEGF >

SEQ ID NO: 9 pocket-heavy chain of < EGFR > scFab with CH1-CL exchange and C-terminal fusion

<VEGF>

SEQ ID NO: 10 knot-heavy chain of < IGF-1R > scFab with C-terminal fusion < Ang-2>

Brief Description of Drawings

FIG. 1 is a schematic structure of a full-length antibody specifically binding to a first antigen 1 without a CH4 domain,

it has two pairs of light chains comprising variable and constant domains in typical order.

FIG. 2a schematic structure of four possible single chain Fab fragments that specifically bind to an antigen.

FIG. 2b schematic structure of a single chain Fv fragment that specifically binds to an antigen.

FIGS. 3a-d schematic structures of different tri-or tetra-specific antibodies according to the invention, characterized in that

In the full-length antibody light/heavy chain of an antibody that specifically binds a second antigen

VL/VH Domain and/or CL/CH1 Domain substitutions (CH3 Domain with no and additional insertion pockets)

Modified).

FIG. 4a identifies Angiopoietin (Angiopoietin) -2, VEGF-A, and,

Schematic structure of tetraspecific antibodies to EGFR and IGF-1R, which are tetravalent

And disulfide-stabilized single-chain Fv fragments as antigen-binding peptides (practice)

Example 1).

FIG. 4b identifies angiopoietin-2, VEGF-A, EGFR and IGF-1R according to the invention

Schematic structure of the tetraspecific antibody of (a), which is tetravalent and uses a single chain Fab

Fragments were used as antigen binding peptides (example 1).

FIG. 5a Tri-specific recognition of angiopoietin-2, VEGF-A and EGFR according to the present invention

Schematic structure of a schematic antibody, which is tetravalent and stabilized with a disulfide

Chain Fv fragments were used as antigen binding peptides (example 2).

FIG. 5b Tri-specific recognition of angiopoietin-2, VEGF-A and EGFR according to the present invention

Schematic structure of an exemplary antibody, which is tetravalent and uses a single-chain Fab fragment as

Antigen binding peptides (example 2).

FIG. 6 Tri-specific recognition of angiopoietin-2, VEGF-A and EGFR according to the present invention

Schematic structure of a schematic antibody, which is trivalent and stabilized with a disulfide

Chain Fv fragments were used as antigen binding peptides (example 3).

FIG. 7 four specificities for identifying EGFR, IGF-1R, c-Met and HER3 in accordance with the present invention

Chain Fv fragments act as antigen binding peptides.

FIG. 8 identifies angiopoietin-2, VEGF-A, EGFR and IGF-1R according to the invention

Size of tetraspecific antibody on high-load 26/60Superdex 200 column

Exclusion chromatography, which is tetravalent and uses single chain Fab fragments as antigen binding

Peptide (example 1).

FIG. 9 identifies angiopoietin-2, VEGF-A, EGFR and IGF-1R according to the invention

SDS-PAGE analysis of the tetraspecific antibodies of (a) under native and denaturing conditions,

which is tetravalent and uses single-chain Fab fragments as antigen-binding peptides (examples)

1)。

FIG. 10 Tri-specific recognition of angiopoietin-2, VEGF-A and EGFR according to the present invention

Size exclusion chromatography of sex antibodies on a high-loaded 26/60Superdex 200 column,

2)。

FIG. 11 is a trispecific for recognition of angiopoietin-2, VEGF-A and EGFR according to the present invention

SDS-PAGE analysis of sex antibodies in native and denatured conditions, which is tetravalent

And a single chain Fab fragment was used as the antigen binding peptide (example 2).

Examples

Materials and general methods

For general information on the nucleotide sequences of the light and heavy chains of human immunoglobulins see: kabat, E.A., et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health service, National Institutes of Health, Bethesda, MD (1991). Amino acids of an antibody chain are numbered and referred to according to EU numbering (Edelman, g.m., et al., proc.natl.acad.sci.usa 63(1969) 78-85; Kabat, e.a., et al., Sequences of Proteins of immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991)).

Recombinant DNA technology

DNA is manipulated using standard methods, such as Sambrook, j.et al, Molecular Cloning: (ii) an Arabidopsis 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 generated by chemical synthesis. The 600-1800bp long gene segment flanked by unique restriction endonuclease sites was assembled by annealing and ligating oligonucleotides, including PCR amplification, followed by cloning into the pPCRScript (Stratagene) -based pGA4 cloning vector via a designated restriction site such as KpnI/SacI or AscI/PacI. The DNA sequence of the subcloned gene fragments was verified by DNA sequencing. The gene synthesis fragments were ordered in Geneart (Regensburg, Germany) according to the given specifications.

DNA sequencing

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

DNA and protein sequence analysis and sequence data management

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

Expression vector

For the expression of the antibodies described, for transient expression (for example in HEK293EBNA or HEK293-F cells) expression plasmid variants based on cDNA tissues with or without the CMV intron a promoter or on genomic tissues with the CMV promoter are used.

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

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

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

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

-one or more unique restriction sites at the 5' end,

immediate early enhancer and promoter from human cytomegalovirus,

in the case of cDNA tissue, 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 exchange), as cDNA or as genomic tissue with immunoglobulin exon-intron tissue,

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

-one or more unique restriction sites at the 3' end.

Fusion genes comprising antibody chains as described below are generated by PCR and/or gene synthesis and assembled by linking the corresponding nucleic acid segments by known recombinant methods and techniques, e.g., using unique restriction sites in each vector. The subcloned nucleic acid sequences were verified by DNA sequencing. For transient transfection, larger quantities of plasmid were prepared by plasmid preparation (nucleobiondax, Macherey-Nagel) from transformed e.

Cell culture technique

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

The tri-or tetra-specific antibodies were expressed by transient co-transfection of each expression plasmid in adherently grown HEK293-EBNA or in suspension grown HEK29-F cells as described below.

Transient transfection in the HEK293-EBNA System

The expression of the heavy chain and modified heavy chain and corresponding light chain and modified light chain by each expression plasmid (e.g., encoding heavy chain and heavy chain, and corresponding light chain and modified light chain) was performed in a medium supplemented with 10% Ultra Low IgG FCS (fetal calf serum,) 2mM L-GlutamineAnd 250. mu.g/ml GeneticinDMEM (Dulbecco's modified Eagle's medium,) Adherently growing HEK293-EBNA cells cultured in (human embryonic kidney cell line 293 expressing the nuclear antigen of EB virus; american type culture Collection deposit number ATCC # CRL-10852, Lot.959218) to express tri-or tetra-specific antibodies. For transfection, FuGENE was used at 4: 1 (range 3: 1 to 6: 1)^TMFuGENE was used for the ratio of reagent (. mu.l) to DNA (. mu.g)^TM6 transfection reagent (Roche Molecular Biochemicals). Proteins were expressed from each plasmid using a molar ratio of (modified and wild type) light chain and heavy chain encoding plasmids of 1: 1 (equimolar) (range 1: 2 to 2: 1), respectively. Day 3 cells were fed L-glutamine ad 4mM, glucose [ Sigma ]]And NAACell culture supernatants containing tri-or tetra-specific antibodies were harvested by centrifugation 5 to 11 days post transfection and stored at-20 ℃. General information on recombinant expression of human immunoglobulins in, for example, HEK293 cells is found in Meissner, P.et al, Biotechnol.Bioeng.75(2001) 197-203.

Transient transfection in the HEK293-F System

The tri-or tetra-specific antibodies were generated by transient transfection of each plasmid (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 instructions of the manufacturer. Briefly, four expression plasmids and 293fectin were used^TMOr transfection of mixtures of fectins (Invitrogen) in Shake flasks or in stirred fermentors in serum-free FreeStyle^TM293 expression Medium (Invitrogen) HEK293-F cells (Invitrogen) grown in suspension. For 2L shake flasks (Corning), HEK293-F cells were seeded at a density of 1.0E 6 cells/mL in 600mL and 8% CO at 120rpm₂And (4) incubation. The following day, cells were transfected with approximately 42mL of mixture A) 20mL of Opti-MEM (Invitrogen) with 600 μ g equimolar ratio of total plasmid DNA encoding the heavy or modified heavy chain and the corresponding light chain (1 μ g/mL) and B)20mL of Opti-MEM +1.2mL 293 or fectin (2 μ l/mL) at a cell density of approximately 1.5E × 6 cells/mL. The glucose solution is 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 purified antibodies and derivatives was determined by measuring the Optical Density (OD) at 280nm using the molar extinction coefficient calculated from the amino acid sequence according to Pace, et al, Protein Science, 1995, 4, 2411-1423.

Determination of antibody concentration in supernatant

Evaluation of cell culture by immunoprecipitation with protein A agarose beads (Roche)Concentration of antibodies and derivatives in the nutrient supernatant. mu.L of protein A agarose beads were washed three times in TBS-NP40(50mM Tris, pH7.5, 150mM NaCl, 1% Nonidet-P40). Subsequently, 1-15mL of cell culture supernatant was applied to protein a agarose beads pre-equilibrated in TBS-NP 40. After 1 hour incubation at room temperature, the beads were washed once with 0.5mL TBS-NP40, twice with 0.5mL 2 Xphosphate buffered saline (2xPBS, Roche) and four times with 0.5mL100mM sodium citrate pH 5, 0 briefly on an Ultrafree-MC-filter column (Amicon). By adding 35. mu.lLDS sample buffer (Invitrogen) to elute bound antibody. Half of the sample was separately mixed withThe sample reducing agents were combined or left unreduced and heated at 70 ℃ for 10 min. Therefore, 5-30. mu.l was applied to 4-12%Bis-Tris SDS-PAGE (Invitrogen) (MOPS buffer for non-reducing SDS-PAGE, bandsAntioxidant MES buffer of running buffer additive (Invitrogen) was used to reduce 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 applied to 200mM KH on an Agilent HPLC 1100 system₂PO₄100mM sodium citrate, Applied Biosystems Poros A/20 column pH 7.4, and eluted from the matrix with 200mM NaCl, 100mM citric acid, pH 2, 5. Eluted protein was quantified by UV absorbance and peak area integration. Purified standard IgG1 antibody served as the standard.

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

Protein purification

Proteins were purified from filtered cell culture supernatant according to standard protocols. Briefly, the antibody was applied to a protein a Sepharose column (GE healthcare) and washed with PBS. Antibody elution was achieved at pH 2.8, followed by immediate neutralization of the sample. Aggregated proteins were separated from monomeric antibodies by size exclusion chromatography (Superdex 200, GEHealthcare) in PBS or in 20mM histidine, 150mM NaCl pH 6.0. Monomeric antibody fractions are pooled, concentrated (when necessary) using, for example, a MILLIPORE AMICONultra (30MWCO) centrifugal concentrator, frozen and stored at-20 ℃ or-80 ℃. Portions of the sample are provided for subsequent protein analysis and analytical characterization, for example by SDS-PAGE, Size Exclusion Chromatography (SEC), or mass spectrometry.

SDS-PA GE

Used according to the instructions of the manufacturerPre-Cast gel system (Invitrogen). In particular, 10% or 4-12% is usedBis-TRIS Pre-Cast gel (pH 6.4) andMES (reduced gels, havingAntioxidant running buffer additive) or MOPS (non-reducing gel) running buffer.

Analytical size exclusion chromatography

Size Exclusion Chromatography (SEC) for determination of the aggregation and oligomerization status of the antibodies was performed by HPLC chromatography. Briefly, protein A purified antibody was applied to 300mM NaCl, 50mM KH on an Agilent HPLC 1100 System₂PO₄/K₂HPO₄Tosoh TSK gel G3000SW column at pH7.5 or Superdex 200 column in 2 × PBS on a Dionex HPLC system (GE Healthcare). Eluted protein was quantified by UV absorbance and peak area integration. The BioRad gel filtration Standard 151-.

Mass spectrometry

The total mass of deglycosylation of the exchange-type antibody was determined and verified by electrospray ionization mass spectrometry (ESI-MS). Briefly, 100. mu.g of purified antibody was treated with 50mU of N-glycosidase F (PNGaseF, Prozyme) at 100mM KH at protein concentrations up to 2mg/ml₂PO₄/K₂HPO₄Deglycosylation at 37 ℃ for 12-24h at pH7, followed by desalting by HPLC on a Sephadex G25 column (GE Healthcare). The mass of each heavy and light chain 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-HCl, followed by desalting. Is provided withThe total mass and the mass of the reduced heavy and light chains were determined by ESI-MS on a Q-Star Elite MS system from the source.

IGF-1R, EGFR, HER3 and c-Met ECD Biacore

The binding of the generated antibodies to human IGF-1R, EGFR, HER3 and c-Met ECD (extracellular domain) was investigated by surface plasmon resonance using a BIACORE T100 instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, goat anti-human IgG, JIR 109-. Binding was measured in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween 20, ph 7.4) at 25 deg.C. ECD (R) from c-Met, IGF-1R or EGFR was added to the solution at various concentrations&D Systems or internal purification). Binding was measured by ECD injection for 80 seconds to 3 minutes; dissociation was measured by washing the chip surface with HBS buffer for 3-10 min, and K was estimated using the 1: 1Langmuir binding model_DThe value is obtained. Due to the low loading density and capture level, monovalent ECD binding is achieved. Negative control data (e.g., buffer curve) was subtracted from the sample curve to correct for in-system baseline drift and reduce noise signals. Sensorgrams and technical affinity data were analyzed using Biacore T100 evaluation software version 1.1.1. Figure 11 shows a schematic of the Biacore assay.

ANGPT2 and VEGF bind BIACORE

Binding of the generated antibodies to human ANGPT2 and VEGF was also investigated by surface plasmon resonance using BIACORE T100 instrument (GE Healthcare Biosciences AB, Uppsala, Sweden). Briefly, for affinity measurements, goats were immobilized on CM5 or CM4 chips via amine coupling<hIgG-Fcg>Antibodies were polyclonal to present antibodies against human ANGPT2 and VEGF. Binding was measured in HBS buffer (HBS-P (10mM HEPES, 150mM NaCl, 0.005% Tween 20, ph 7.4) with or without 5mM Ca2 +. 25 ℃ purified ANGPT2-His or VEGF165/VEGF121-His (R) was added to the solution at various concentrations&D Systems or internal purification). Passing 3 min ANGPT2/VEGF injection to measure binding; dissociation was measured by washing the chip surface with HBS buffer for 3-5 min, and K was estimated using the 1: 1Langmuir binding model_DThe value is obtained. Negative control data (e.g., buffer curve) was subtracted from the sample curve to correct for in-system baseline drift and reduce noise signals. Sensorgrams and technical affinity data were analyzed using Biacore T100 evaluation software version 1.1.1. Simultaneous binding in BIACORE

Simultaneous binding of tetra-and tri-specific antibodies to EGFR, IGF-1R, Ang-2 and VEGF or EGFR, IGF-1R, HER3 and c-Met or EGFR, Ang-2 and VEGF, respectively.

Binding in the tetra-or tri-specific antibody format was compared to the binding of the derivatized binding module and the 'wild-type' IgG of the bispecific antibody. These analyses were performed by applying surface plasmon resonance (Biacore) as described above. To demonstrate simultaneous binding, binding characteristics were analyzed by Surface Plasmon Resonance (SPR) using a Biacore T100 instrument (Biacore AB, Uppsala).

Anti-human IgG antibodies for capture were immobilized on the surface of CM5 biosensor chip using amine coupling chemistry. The flow cell was activated with a 1: 1 mixture of 0.1M N-hydroxysuccinimide and 0.1M 3- (N, N-dimethylamino) propyl-N-ethylcarbodiimide at a flow rate of 5. mu.l/min. Anti-human IgG antibodies were injected at 10. mu.g/ml in sodium acetate, pH 5.0, which resulted in a surface density of about 12000 RU. The reference control flow cell was treated with antibody in the same manner but with the vehicle buffer only instead of capture. The surface was blocked by injection with 1M ethanolamine/HCl, pH 8.5. Multispecific antibodies were diluted in HBS-P and injected at a flow rate of 5. mu.l/min. For antibody concentrations between 1 and 50nM, the contact time (binding phase) is 1 min. EGFR/IGF-1R/HER3/c-Met-ECD and Ang-2 or VEGF, respectively, were injected at increasing concentrations. All interactions were performed at 25 ℃ (standard temperature). After each binding cycle a regeneration solution was injected at a flow of 5 μ l/min for 60sec of 3M magnesium chloride to remove any non-covalently bound proteins. The signals are detected at a rate of one signal per second. Samples were injected at increasing concentrations.

Example 1: generation, expression, purification and characterization of tetraspecific and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, EGFR and IGF-1R

In the first instance, by passing through (G)₄S)₄Linker fusion of disulfide stabilized scFv against EGFR and scFv against IGF-1R to the C-terminal portions of the first and second heavy chains of CH1/CL (C kappa) domain exchange antibodies with a knob-in-hole recognizing angiopoietin-2 and VEGF with its variable domains, respectively, generated tetraspecific and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, EGFR and IGF-1R (fig. 4 a). The sequences of the 4 antibody chains are shown in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 4.

key data
		Expression (yield) -mg/mL	14，5
Purification (protein A homogeneity) -%)	91，3
		Yield after SEC-mg/mL	10，4
Homogeneity-after preparative SEC%	99，7

In a second example, by passing through (G)₄S)₂Linkers fused scFab against EGFR and scFab against IGF-1R to the C-terminal portions of the first and second heavy chains, respectively, of CH1/CL (C kappa) domain exchange antibodies with a knob-in-hole that recognize angiopoietin-2 and VEGF with its variable domains, generating tetraspecific and tetravalent antibodies that recognize angiopoietin-2, VEGF-A, EGFR and IGF-1R (FIG. 4 b). The sequences of the 4 antibody chains are shown in SEQ ID NOs: 1. SEQ ID NO: 5. SEQ ID NO: 3 and SEQ ID NO: 6.

key data
		Expression (yield) -mg/mL	12，2
Purification (protein A homogeneity) -%)	74，4
		Yield after SEC-mg/mL	6，8
Homogeneity-after preparative SEC%	98，4

In another example similar to the second example, the first and second phases are separated by a filter (G)₄S)₂Linker fusion of scFab against EGFR and scFab against IGF-1R to recognition of angiogenesis by its variable domain with knob-in-hole, respectivelyThe CH1/CL (C kappa) domains of adult-2 and VEGF were exchanged for the C-terminal portions of the second and first heavy chains of the antibody, generating a tetraspecific and tetravalent antibody that recognizes angiopoietin-2, VEGF-A, EGFR and IGF-1R (similar to fig. 4b, but with the scFab against IGF-1R fused to the junction ANG2 binding heavy chain and the scFab against EGFR fused to the hole VEGF binding heavy chain). The sequences of the 4 antibody chains are shown in SEQ ID NOs: 1. SEQ ID NO: 3. SEQ ID NO: 9 and SEQ ID NO: 10.

these antibody variants were generated by classical molecular biology techniques as described in the general methods section above and transiently expressed in HEK293F cells as described above. Subsequently, they were purified from the supernatant by a combination of protein a affinity chromatography and size exclusion chromatography. The obtained products were characterized for identity (by mass spectrometry) and analytical properties such as purity (by SDS-PAGE), monomer content and stability (FIGS. 8-9, based on SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 9 and SEQ ID NO: 10).

The (simultaneous) binding of the four antibodies specifically to the four overlaid antigens (angiopoietin-2, VEGF-A, EGFR and IGF-1R) was shown by Biacore using the method described above.

Table:based on SEQ ID NO: 1. SEQ ID NO: 3. SEQ ID NO: 9 and SEQ ID NO: binding of tetraspecific and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, EGFR and IGF-1R.

Analyte	k_a(1/Ms)	k_d(1/s)	K_D(nM)
				EGFR(HER1)	3.1E+05*	3.9E-05*	12.8*
IGF-1R			Low binding affinity
				Ang-2	n.d.***	n.d.***	138***
VEGF	5.0E+04*	＜1E-06*	＜1E-11*

Capture by anti-human antibodies

(ii) capture via HER1

Ang-2 surface

Example 2: generation, expression, purification and characterization of trispecific and tetravalent antibodies recognizing angiopoietin-2, VEGF-A and EGFR

In the first instance, by passing through (G)₄S)₄Linker fusion of disulfide stabilized scFv against EGFR to two CH1/CL (C kappa) Domain exchange antibodies with a knob-in-hole recognizing angiopoietin-2 and VEGF with its variable domainThe C-terminal part of the heavy chain produced tri-and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, EGFR and IGF-1R (FIG. 5 a). The sequences of the 4 antibody chains are shown in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 7.

key data
		Expression (yield) -mg/mL	20，1
Purification (protein A homogeneity) -%)	64，1
		Yield after SEC-mg/mL	12，0

Homogeneity-after preparative SEC%

100

Table:according to FIG. 5a, the binding of tri-and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, and EGFR.

Binding affinity	k_a(1/Ms)	k_d(1/s)	K_D(nM)
				EGFR(HER1)	4.7E+04	2.3E-04	6
hAng-2	1E+06	1.7E-04	0.2
				hVEGF	1E+05	＜1E-06	＜0.1

In a second example, by passing through (G)₄S)₂The linker fused the two scfabs to the C-terminal parts of the two heavy chains of a CH1/CL (C kappa) domain exchange antibody with a knob-in-hole recognizing angiopoietin-2 and VEGF with its variable domain, generating tri-and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, EGFR and IGF-1R (fig. 5 b). The sequences of the 4 antibody chains are shown in SEQ ID NO: 1.SEQ ID NO: 5. SEQ ID NO: 3 and SEQ ID NO: 9.

these antibody variants were generated by classical molecular biology techniques as described in the general methods section above and transiently expressed in HEK293F cells as described above. Subsequently, they were purified from the supernatant by a combination of protein a affinity chromatography and size exclusion chromatography. The obtained products were characterized for identity (by mass spectrometry) and analytical properties such as purity (by SDS-PAGE), monomer content and stability (FIGS. 10-11, based on SEQ ID NO: 1, SEQ ID NO: 5, SEQ ID NO: 3 and SEQ ID NO: 9).

The (simultaneous) binding of the four antibodies specifically to the three overlaid antigens (angiopoietin-2, VEGF-a and EGFR) was shown by Biacore using the method described above.

Table:according to FIG. 5b, the binding of tri-and tetravalent antibodies recognizing angiopoietin-2, VEGF-A, and EGFR.

Analyte	k_a(1/Ms)	k_d(1/s)	K_D(nM)
				EGFR(HER1)	3.7E+04*	3.4E-04*	2.7*
Ang-2	n.d.**	n.d.**	176**
				VEGF	6.7E+04*	＜1E-06*	＜0.01*

Capture by anti-human antibodies

Ang-2 surface

Example 3: production, expression, purification and characterization of trispecific and trivalent antibodies recognizing angiopoietin-2, VEGF-A and EGFR

In the first instance, by passing through (G)₄S)₄The linker fuses a disulfide stabilized scFv against EGFR to the C-terminal part of two heavy chains of a CH1/CL (C kappa) domain exchange antibody with a knob-in-hole, recognizing angiopoietin-2 and VEGF with its variable domain, generating a trispecific and trivalent antibody recognizing angiopoietin-2, VEGF-A, EGFR and IGF-1R (fig. 6). The sequences of the 4 antibody chains are shown in SEQ ID NOs: 1. SEQ ID NO: 2. SEQ ID NO: 3 and SEQ ID NO: 8.

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

Key data
		Expression (yield) -mg/mL	40，9
Purification (protein A homogeneity) -%)	77，3
		Yield after SEC-mg/mL	22，3
Homogeneity-after preparative SEC%	100

Claims

1. A trispecific antibody comprising:

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

c) wherein an antigen binding peptide that specifically binds to another antigen is fused to the C-terminus of the heavy chain of a) or b) via a peptide linker, wherein the peptide linker is (G)_xS)_nOr (G)_xS)_nG_mWherein G ═ glycine, S ═ serine, x ═ 3, n ═ 3, 4, 5, or 6 and m ═ 0, 1, 2, or 3, x ═ 4, n ═ 2, 3, 4, or 5 and m ═ 0, 1, 2, or 3, the antigen binding peptide is selected from the group consisting of: scFv fragments and scFab fragments, and;

a) the CH3 domain of the heavy chain of the full-length antibody of b) and the CH3 domain of the modified heavy chain of the full-length antibody of b) meet at an interface that constitutes the initial interface between the CH3 domains of said antibody; wherein the interface is altered to facilitate formation of the trispecific antibody, wherein the alteration is characterized by:

i) altering the CH3 domain of one heavy chain such that within the initial interface of the CH3 domain of one heavy chain meeting the initial interface of the CH3 domain of another heavy chain in the trispecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the interface of the CH3 domain of one heavy chain that can locate in a cavity within the interface of the CH3 domain of another heavy chain;

and is

ii) altering the CH3 domain of the other heavy chain such that within the initial interface of the first CH3 domain in the second CH3 domain encountered in the trispecific antibody, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain wherein the protuberance within the interface of the first CH3 domain can be located.

2. A trispecific antibody comprising:

c) wherein two antigen binding peptides that specifically bind to another antigen are fused to the C-terminus of the heavy chains of a) and b) via a peptide linker, wherein the peptide linker is (G)_xS)_nOr (G)_xS)_nG_mWherein G ═ glycine, S ═ serine, x ═ 3, n ═ 3, 4, 5, or 6 and m ═ 0, 1, 2, or 3, x ═ 4, n ═ 2, 3, 4, or 5 and m ═ 0, 1, 2, or 3, said compound being useful as a medicament for treating cardiovascular diseasesThe antigen binding peptide is selected from the group consisting of: scFv fragments and scFab fragments, and;

and is

3. Antibody according to claim 2, characterized in that it comprises under c) two identical antigen binding peptides specifically binding to a third antigen, selected from the group consisting of: scFv fragments and scFab fragments.

4. A tetraspecific antibody comprising:

c) wherein two antigen binding peptides that specifically bind two additional antigens are fused to the C-terminus of the heavy chains of a) and b) via a peptide linker, wherein the peptide linker is (G)_xS)_nOr (G)_xS)_nG_mWherein G ═ glycine, S ═ glycineSerine, where x is 3, n is 3, 4, 5 or 6 and m is 0, 1, 2 or 3, where x is4, n is 2, 3, 4 or 5 and m is 0, 1, 2 or 3, said antigen binding peptide being selected from the group consisting of: scFv fragments and scFab fragments, and;

a) the CH3 domain of the heavy chain of the full-length antibody of b) and the CH3 domain of the modified heavy chain of the full-length antibody of b) meet at an interface that constitutes the initial interface between the CH3 domains of said antibody; wherein the interface is altered to facilitate formation of the tetraspecific antibody, wherein the alteration is characterized by:

i) altering the CH3 domain of one heavy chain such that within the initial interface of the CH3 domain of one heavy chain meeting the initial interface of the CH3 domain of another heavy chain in the tetraspecific antibody, an amino acid residue is replaced with an amino acid residue having a larger side chain volume, thereby creating a protuberance within the interface of the CH3 domain of one heavy chain that can locate in a cavity within the interface of the CH3 domain of another heavy chain;

and is

ii) altering the CH3 domain of the other heavy chain such that within the initial interface of the first CH3 domain in the second CH3 domain encountered in the tetraspecific antibody, amino acid residues are replaced with amino acid residues having a smaller side chain volume, thereby generating a cavity within the interface of the second CH3 domain wherein the protuberance within the interface of the first CH3 domain can be located.

5. The antibody according to any one of claims 1 to 4, characterized in that the antigen binding peptide is a scFv fragment.

6. The antibody according to any one of claims 1 to 4, characterized in that the antigen binding peptide is a scFab fragment.

7. The antibody according to any one of claims 1 to 4, characterized in that the amino acid residue with a larger side chain volume is selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), and 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).

8. Antibody according to claim 5, characterized in that the amino acid residues with larger side chain volume are selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), and 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).

9. Antibody according to claim 6, characterized in that the amino acid residues with larger side chain volume are selected from the group consisting of: arginine (R), phenylalanine (F), tyrosine (Y), tryptophan (W), and 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).

10. The antibody according to any one of claims 1, 2 and 4, characterized in that the two CH3 domains are further altered by introducing cysteine (C) as an amino acid in the corresponding position of each CH3 domain such that a disulfide bridge can be formed between the two CH3 domains.

11. A method for preparing an antibody according to any one of claims 1-10, comprising the steps of:

a) transforming a host cell with a vector comprising a nucleic acid molecule encoding an antibody according to any one of claims 1-10;

c) Recovering the antibody molecule from the culture.

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

13. A composition comprising an antibody according to any one of claims 1 to 10.

14. The composition according to claim 13, which is a pharmaceutical or diagnostic composition.

15. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 10 and at least one pharmaceutically acceptable excipient.