MX2014000816A

MX2014000816A - Multivalent antigen-binding fv molecule.

Info

Publication number: MX2014000816A
Application number: MX2014000816A
Authority: MX
Inventors: Melvyn Little; Fabrice Le Gall
Original assignee: Affimed Therapeutics Ag
Priority date: 2011-07-22
Filing date: 2011-07-22
Publication date: 2014-07-09
Also published as: MX347829B; WO2013013700A1; JP2014527515A; CN103687879A; BR112014001573B8; AU2011373925B2; BR112014001573B1; CA2842649A1; CN103687879B; AU2011373925A1; RU2613368C2; BR112014001573A2; CA2842649C; JP5938473B2; RU2013157040A

Abstract

In one aspect, the present invention relates to an antigen-binding molecule specific for albumin and CD3 comprising two polypeptide chains, each polypeptide chain having at least four variable domains in an orientation preventing Fv formation and the two polypeptide chains are dimerized with one another thereby forming a multivalent antigen-binding molecule. On each of the two polypeptide chains the four variable domains are arranged in the order VLA-VHB-VLB-VHA from the N-terminal to the C-terminal of the polypeptide. Compositions of the antigen-binding molecule and the methods of using the antigen-binding molecule or the compositions thereof for treatment of various diseases are also provided herein.

Description

MULTIVALENT ANTIGEN FV MOLECULE FV FIELD OF THE INVENTION The invention relates to novel cascade Fv diabodies and uses thereof.

Several formats of multivalent recombinant antibody fragments have been designed as alternatives to antibodies derived from the quadroma.

US 7,129,330, Kripiyanov et al. J. Mol. Bíol. (1999) 293, 41-56 and Kripiyanov Meth. Mol. Biol. (2009) 562, 177-193 describe the construction and production of a particular format of multivalent antibody fragments which are called "tandem diabodies" or connected online (TandAb®), since their design is based on the intermolecular pairing of the VH and VL variable domains of two different polypeptides as described for diabodies (Holliger et al., 1993, Proc Nati Acad Sci USA, 90: 6444-6448). The antibodies described are bispecific for CD19 and CD3. In contrast to the bivalent scFy-scFv (scFv) 2 tandems, the tandem or in-line diabodies are tetravalent, because they have four antigen-binding sites. Polypeptides with the order of domain VHA-VLB-VHB-VLA are described from N-terminal to C-terminal of the polypeptides that form the tandem diabodies. The orders of the variable domains and the binding peptides therebetween were designed so that each domain is associated with a complementary domain in another identical molecule thereby forming the dimerized tetravalent tandem diabodies. The tandem diabodies are devoid of constant immunoglobulin domains. It was reported that tandem diabodies have advantages such as high affinity, greater avidity, lower elimination rates and exhibit an efficient in vitro and a favorable live.

Several additional tandem diabodies are known that comprise antibody specificities such as, for example, anti-CD16, anti-EpCAM and anti-CD30. In all cases, however, the order of the four antibody domains along the polypeptide chains of tandem diabodies from N-terminal to C-terminal was always VHA-VLB-HBV-VLA where VH and VL represent the heavy and light chain variable domains of the antibody antibody with specificities for antigens A and B respectively.

These bispecific tandem diabodies can form a bridge between a tumor cell (eg, B-CLL cell) and an effector cell of the human immune system (NK cell, T cell, monocyte, macrophage or granulocyte) thus allowing the elimination of the tumor cell. The strong union of the tumor cell and the cytotoxic cell induces the destruction of the tumor cell. Although such tandem or online antibodies have proven favorable for therapeutic applications, for example for therapeutic concepts for the treatment of tumors, there remains a need for improved antigen binding molecules.

BRIEF DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a dimeric antigen binding molecule comprising a first and a second polypeptide chain, each of the first and second polypeptide chains comprising (a) a first VLA domain that is a light chain variable domain specific for a first antigen A; (b) a second VHB domain that is a heavy chain variable domain specific for a second B antigen; (c) a third VLB domain that is a light chain variable domain specific for the second antigen B; and (d) a fourth VHA domain that is a heavy chain variable domain specific for the first antigen A, where the domains are arranged in each of the first and second polypeptide chains in the order of VLA-HBV-VLB-VHA of the N-terminal to the C-terminal of the polypeptide chains, and the first VLA domain of the first polypeptide chain is associated with the fourth VHA domain of the polypeptide chain to form an antigen binding site for the first antigen A; and the second HBV domain of the first polypeptide chain is associated with the third VLB domain of the second polypeptide chain to form an antigen binding site for the second B antigen; and the third VLB domain of the first polypeptide chain is associated with the second VHB domain of the second polypeptide chain to form an antigen binding site for the second B antigen; and the fourth VHA domain of the first polypeptide chain is associated with the first VLA domain of the second polypeptide chain to form an antigen binding site for the first antigen A.

In some embodiments, the antigen binding molecule as described herein is a homodimer and the first and second polypeptide chains have the same amino acid sequence. In some embodiments, the first and second polypeptide chains are associated non-covalently. In some embodiments, the antigen-binding molecule is tetravalent. In some embodiments, the antigen-binding molecule is bispecific. In some embodiments, the domains are human domains or humanized domains. In some embodiments, the antigen-binding molecule comprises at least one additional functional unit. In some modalities, the molecule of Antigen binding is specific for a B cell, T cell, Natural Killer (NK) cell, myeloid cell or phagocytic cell. In some embodiments, the antigen binding molecule is bispecific, whose antigen binding molecule is also specific for a tumor cell. In some embodiments, the first light chain variable domain (VLA) and the first heavy chain variable domain (VHA) are specific for a tumor cell. In some embodiments, the antigen-binding molecule is bispecific for albumin and CD3.

In another aspect, the present invention provides a polypeptide chain comprising (a) a first VLA domain that is a light chain variable domain specific for a first antigen A; (b) a second VHB domain that is a heavy chain variable domain specific for a second B antigen; (c) a third VLB domain that is a light chain variable domain specific for a second B antigen; and (d) a fourth HAV domain which is a heavy chain variable domain specific for the first antigen A; where the domains are arranged in the polypeptide chain in the order VLA-VHB-VLB-VHA from the N-terminal to the C-terminal of the polypeptide chains. In some embodiments, the first VLA domain and the fourth VHA domain do not associate to form an antigen binding site for the first antigen A and the Second VHB domain and the third VLB domain do not associate to form an antigen binding site for the second antigen B. In some embodiments, the first VLA domain and the second VHA domain, the second VHB domain and the third VLB domain and the third VLB domain, and the fourth VHA domain are separated by no more than about 12 amino acid residues. In some embodiments the polypeptide chain comprises amino acid residues upstream of the first VLA domain and / or downstream of the fourth VHA domain. In some embodiments, the polypeptide chain is linked or linked to a further functional unit. In a particular embodiment, the variable domains are specific for albumin and CD3.

In another aspect, the present invention provides a nucleic acid molecule that encodes a polypeptide chain as described herein. In another aspect, the present invention provides a pharmaceutical composition comprising the antigen-binding molecule, the polypeptide chain or the nucleic acid molecule as described herein and a pharmaceutically acceptable carrier.

In another aspect, the present invention provides a medical use of the antigen binding molecule as a medicament for the treatment of an autoimmune disease, inflammatory disease, disease infectious, allergic, cancer and / or as an immunosuppressive drug.

BRIEF DESCRIPTION OF THE FIGURES Figure 1 illustrates the genetic organization of a construct encoding an antigen molecule according to the invention, where VLA represents a light chain variable immunoglobulin domain specific for an antigen A, HBV represents a heavy chain variable immunoglobulin domain specific for an antigen B, VLB represents a light chain variable immunoglobulin domain specific for an antigen B, HAV represents a heavy chain variable immunoglobulin domain specific for an antigen A, a peptide linker or a peptide bond connecting VLA and HBV, L2 a peptide linker or a peptide bond that connects HBV and VLB, and L3 a peptide linker or peptide bond that connects VLB and VHA.

Figures 2A, 2B and 2C illustrate the formation of a dimeric antigen binding molecule according to the invention from non-functional monomeric polypeptide chains (A) by intramolecular pairing of variable domains of a first polypeptide chain 1 and a second chain polypeptide 2 with another (B) to a functional antigen binding molecule according to the invention in the format of a tandem diabody or connected on line where "1" represents the first polypeptide chain, "2" represents the second polypeptide chain, VLA represents a light chain variable immunoglobulin domain specific for an A antigen, HBV represents a specific heavy chain variable immunoglobulin domain for a B antigen, VLB represents a light variable chain immunoglobulin domain specific for the B antigen, HAV represents a heavy chain variable immunoglobulin domain specific for the A antigen, a peptide linker or a peptide bond connecting VLA and HBV, L2 a peptide linker or a peptide bond that connects HBV and VLB, and L3 a peptide linker or peptide bond that connects VLB and VHA.

Figure 3 shows a comparison of the tandem diabodies or connected in CD19xCD3 lines in a cytotoxicity assay. Option 0 = Al antibody with the domain order VHA-VLB-VHB-VLA. Option 2 = antibody B with the domain order VLA-VHB-VLB-VHA according to the invention. LxlO4 calcein-labeled Raji cells were incubated with 5xl05 of PBMC in the presence of increasing concentrations of the indicated CD19xCD3 tandem diabodies. The PBMC were cultured overnight in the presence of 25 U / mL of human IL-2 before they were used as effector cells in the assay. After 4h of incubation, the fluorescent calcein in the culture medium Cell liberated from the apoptotic target cells was measured at 520 nm and the% specific lysis was calculated. The CE5o values were analyzed by non-linear regression using the GraphPad software. The mean and standard deviations of duplicates were plotted.

Figure 4 shows a comparison of CD19xCD3 tandem diabodies in a cytotoxicity assay. Option 0 = A2 antibody with the domain order VHA-LB-VHB-VLA. Option 2 = C antibody for the purpose VLA-VHB-VLB-VHA domain according to the invention. Lx104 Ra i cells labeled with calcein were incubated with 5xl05 freshly isolated from PBMC in the presence of increasing concentrations of the indicated CD19xCD3 tandem diabodies. After 4h of incubation the fluorescent calcein in the cell culture medium released from apoptotic target cells was measured at 520 nm and the% specific lysis was calculated. The EC50 values were analyzed by non-linear regression using the GraphPad software. The mean and the standard deviation of duplicates were plotted.

Figure 5 shows the modulation of TCR by diabodies HSAxCD3 TandAb of Example 2 in the presence or absence of HSA. Jurkat CD3 + cells were cultured for 2 h in the presence of increasing concentrations of antibodies HSAxCD3 TandAb option 0 (VHA-VLB-VHB-VLA; triangle) or option 2 (VLA-VHB-VLB-VHA according to the invention; square) with antibodies (filled symbols) or without (open symbols) adding 50 mg / mL of HSA. After washing, the remaining TCR / CD3 complexes were measured by flow cytometry using a conjugated anti-TCRa / p-PC5 antibody. The mean fluorescence values were used for the non-linear regression analysis (CAB-306 experiment).

Figure 6 shows the vector map with the restriction sites of pCDNA5FRT coding for antibody B. VH and VL: variable domains of heavy and light chains.

Figure 7 shows the vector map with the restriction sites of pSKK3 encoding antibody C. VH and VL: variable domains of heavy and light chains.

DETAILED DESCRIPTION OF THE INVENTION In one aspect, the present invention provides a recombinant dimeric and tetravalent antigen binding molecule with four immunoglobulin domains (two heavy chain variable domains and two light chain variable domains) linked to each other in a polypeptide chain and arranged in the order VLA-VHB-VLB-VHA from the N-terminal to the C-terminal of the polypeptide chain. That antigen-binding molecule of the present invention activates an improved biological activity, as, for example, an improved immune response or improved immune suppression.

In an embodiment, this illustrates that a bispecific dimeric antibody binding molecule of the tandem diabody format that is specific for CD3 and CD19 and that has polypeptide chains with the order of domain VLA-HBV-VLB-VHA is more than 60 times more active in vitro, ie cytotoxic, than a corresponding tandem diabody molecule with the same domains, but in the inverted domain order VHA-VLB-HBV-VLA.

In another embodiment, this illustrates that a dimeric antigen binding molecule, bispecific of the tandem diabody format that is specific for an albumin (HSA) and CD19 and having polypeptide chains with the order of domain VLA-VHB-VLB-VHA it has a significantly more effective T cell receptor modulation activity in vitro, that is, it is more immunosuppressive than a corresponding tandem diabody molecule with the same domains, but in the inverted domain order VHA-VLB-HBV-VLA.

Thus, tandem diabodies with the order of domain VLA-HBV-VLB-VHA from the N-terminal to the C-terminal of the polypeptide chains have a greater potential for immunotherapy. One more advantage of the improved biological activity is that the therapeutic doses effective for these tandem diabodies can be reduced. In addition, the side effects caused by the antigen binding molecules administered can also be reduced due to the lower doses. Without being bound by any theory, the new domain order allows a modified crosslinking of the dimeric antigen binding molecule between antigen A and antigen B compared to the tandem diabodies of the art and, in certain aspects of the invention , this will allow the molecule to bind target antigens, eg, receptors, more efficiently than the dimeric antigen binding molecules of the art.

Therefore, the biological activity of an antigen, dimeric binding molecule, such as a tandem diabody can be improved, when the four variable domains of each polypeptide chain forming the dimer antigen binding molecule are arranged in the order VLA -VHB-VLB-VHA from the N-terminal to the C-terminal of each polypeptide chain. Activated "biological activity" depends on the specificities of the antigen binding molecule and may encompass cytotoxicity, phagocytosis, antigen presentation, cytokine release or immune suppression, eg, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis mediated by antibody-dependent cells (ADCP) and / or Complement-dependent cytotoxicity (CDC). In some embodiments, the present invention provides a dimeric antigen binding molecule comprising a first and a second polypeptide chain, wherein each of the first and second polypeptide chains comprises a first VLA domain that is a light chain variable domain specific for a first antigen A, a second VHB domain that is a heavy chain variable domain specific for a second antigen B, a third domain VLB that is a light chain variable domain specific for the second antigen B, a fourth domain VHA that is a heavy chain variable domain specific for the first antigen A, and the first domains are arranged in each of the first and second polypeptide chains in the order VLA-VHB-VLB-VHA from the N-terminus to the C-terminus of the polypeptide chains .

In some embodiments, the first, second, third and fourth variable domains are arranged in an orientation that avoids intramolecular pairing within the same polypeptide chain and the first polypeptide chain is associated, that is, dimerized, with the second polypeptide chain, so that the first VLA domain of the first polypeptide chain is associated with the fourth VHA domain of the second polypeptide chain to form an antigen binding site for the first antigen A, the second HBV domain of the first polypeptide chain is associated with the third VLB domain of the second polypeptide chain to form an antigen binding site for the second B antigen, the third VLB domain of the first polypeptide chain is associated with the second VHB domain of the second polypeptide chain to form an antigen binding site for the second B antigen and the fourth VHA domain of the first polypeptide chain is associated with the first VLA domain of the second polypeptide chain to form a binding site of antigen for the first antigen A.

The term "antigen binding molecule" refers to an immunoglobulin derivative with multivalent antigen binding properties, preferably having at least four antigen binding sites. Each antigen binding site is formed by a VH heavy chain variable domain and a VL light chain variable domain of the same antigen, i.e., epitope, specificity. Preferably, the antigen-binding molecule according to the invention is devoid of constant immunoglobulin domains or fragments of the constant immunoglobulin domains, but in certain cases described below, a constant domain or parts thereof can be linked to the molecule of antigen binding.

The antigen binding molecule is "dimeric", which term refers to a complex of two polypeptide monomers. These two polypeptide monomers are the first and second polypeptide chains. Preferably, the antigen binding molecule is a "homodimer", which term means the antigen binding molecule is composed of identical polypeptide monomers. In a preferred homodimeric antigen binding molecule according to the invention, the first and second polypeptide chains can have the same amino acid sequence, i.e., that the first and second polypeptide chains are identical and, thus, are encoded and expressed by the same simple polynucleotide. This is different in the case of so-called bispecific diabodies, which are heterodimers that are encoded by two different polynucleotides. In the first case each of the first and second polypetidic chains contain four variable domains, which form four binding sites and the antigen binding molecule is tetravalent. These tetravalent homodimeric antigen binding molecules have received some recognition in the art as tandem diabodies or connected online.

Preferably, the antigen-binding molecule of the first and second polypeptide chains are associated in a non-covalent manner with each other, in particular, with the proviso that there is no covalent bond between the first and second polypeptide chains. However, if desired, the two polypeptide chains can be further stabilized by at least one covalent bond, for example, by a disulfide bond between the cysteine residues of different polypeptide chains.

The term "polypeptide chain" refers to a polymer of residual amino acids linked by amide bonds. The first and second polypeptide chains are preferably single chain fusion proteins which are not branched. In the case of the first and second polypeptide chains the four domains are arranged so that the second VHB domain is C-terminal from the first VLA domain, the third VLB domain is C-terminal from the second VHB domain and the fourth VHA domain is C-terminal from the third VLB domain. The first and second polypeptide chains can have contiguous residual amino acids in addition to being N-terminal towards the first VLA and / or C-terminal domain towards the fourth VHA domain. For example, the polypeptide chain may contain a Tag sequence, preferably at the C-terminus, which may be useful for the purification of the polypeptide. An example of a Tag sequence is His-Tag, for example, a His-Tag consisting of six residues of His.

In some modalities, the first, second, third and fourth domains are connected covalently, so that the domains of the same polypeptide chain do not associate, that is, they mate with each other. The domains can be linked so that the first VLA domain is linked to the second VHB domain by means of a first linker Ll, the second VHB domain is linked to the third VLB domain by means of a second linker L2 and the third domain VLB is linked to the fourth VHA domain by a third linker L3, where the first linker Ll and the third linker L3 are distant relative to the linker L2 in each of the first and second polypeptide chains. The linker Ll, linker L2 and linker L3 can each be a peptide linker comprising at least one residual amino acid or a peptide bond without any intermittent amino acid between the adjacent domains.

In some embodiments, the length of each of the linkers Ll, L2 and L3 is such that the domains of the first polypeptide chain can also be associated with the domains of the second polypeptide chain to form the dimeric antigen binding molecule. The length of the binders influences the flexibility of the antigen binding molecule. The desired flexibility of the molecule Antigen binding depends on the density of the target antigen and the accessibility of the target antigen, that is, the epitopes. The longer binders provide more flexible antigen binding molecules with more agile antigen binding sites. The effect of linker length on the formation of dimeric antigen binding molecules is described, for example, in Todorovska et al., 2001 Journal of Immunological Methods 248: 47-66; Perisic et al., 1994 Structure 2: 1217-1226; Le Gall et al., 2004, Protein Engineering 17: 357-366 and WO 94/13804.

In certain preferred embodiments, the linkers Ll, L2 and / or L3 are "short", ie they consist of 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or approximately 12 residual amino acids. These short linkers favor the correct dimerization of the first and second polypeptide chains by binding them and forming antibody binding sites between the variable domains of light chain and heavy chain variable domains of different polypeptide chains. In particular, the central linker L2 should be short, so as to avoid the formation of a single chain Fv antigen binding unit (scFv) within the same polypeptide chain by the two adjacent VHB and VLB domains. The central linker L2 influences the flexibility of the polypeptide chain. If the central linker L2 is long, and flexible (consisting generally of about 12 or more residual amino acids) the polypeptide chain can be bent from head to tail and form a single chain antigen binding molecule known in the art as a single chain diabody. If the central linker L2 is short and rigid, the polypeptide chain can not bend from head to tail and is dimerized with another polypeptide chain. The number of residual amino acids in a linker to avoid a head-to-tail fold also depends on the type of variable domains combined in the polypeptide. In general, shortening the binder to approximately 12 or fewer residual amino acids generally prevents adjacent domains of the same polypeptide chain from interacting with each other. Therefore, the central linker L2 and the distal linkers Ll and L3 should preferably consist of approximately 12 or less residual amino acids to avoid mating of adjacent domains of the same polypeptide chain. In a preferred embodiment of the invention, the ligands Ll, L2 and / or L3 consist of from about 3 to about 10 contiguous residual amino acids. The binders may consist of different numbers of residual amino acids, but it is preferred that the distal linkers Ll and L3 have the same number of residual amino acids or do not differ in length by more than two. residual amino acids. In a certain aspect of the invention at least one of the ligands Ll, L3 and / or L3 consists of 9 residual amino acids. In a particular embodiment of the invention the three linkers Ll, L2 and L3 consist of 9 amino acid residues. In some embodiments, at least one of the ligands Ll, L2 and / or L3 consist of less than 10 to 3 residual amino acids.

Additional amino acid residues provide extra flexibility. In an alternative aspect the central linker L2 can have approximately 12 or less residual amino acids to avoid the head-to-tail bending of the polypeptide chain and at least one of the distal linkers Ll and / or L3 can have more than about 12 residual amino acids for Avoid extra flexibility. In another embodiment, two polypeptide chains having a central linker L2 with more than 12 residual amino acids are correctly dimerized from each other to a dimeric, tetravalent antigen binding molecule (see for example Le Gall et al., 2004 Protein Engineering 17: 357 -366). However, if longer binders, for example consisting of about 13 or more, in particular of about 15 or more, residual amino acids are used, the dimeric antigen binding molecule can be further stabilized by at least one covalent bond between those two chains polypeptides.

With respect to the amino acid composition of the linkers, in some embodiments, the peptides are selected so as not to interfere with the dimerization of the first and second polypeptide chains. For example, binders comprising glycine and serine residues generally provide flexibility and resistance to the protease. The amino acid sequence of the linkers can be optimized, for example, by phage display methods to improve antigen binding and the production yield of the molecule. In particular embodiments of the invention, the linker may comprise the amino acid sequence GGSGGSGGS.

The first VLA domain, the second VHB domain, the third VLB domain and the fourth VHA domain are light chain and heavy chain variable domains of an immunoglobin. The variable domains comprise the hypervariable loops or complementarity binding regions (CDRs) that contain the residues in contact with the antigen and the segments that contribute to the fold and correct presentation of the CDRs. It is preferred that each of the heavy chain and light chain variable domains comprise the three respective CDRs. The domains can be derived from an immunoglobulin class, for example, IgA, IgD, IgE and IgM or a subclass thereof. The immunoglobulin can be of animal origin, in particular mammalian. Each domain can be a heavy or light chain variable domain of complete immunoglobulin, a mutant, a fragment or derivative of any natural variable domain, or a synthetic one, for example a recombinant domain that is genetically modified. A derivative is a variable domain which differs by the deletion, substitution, addition or insertion of at least one amino acid of the amino acid sequence of a natural variable domain. Synthetic domains, for example recombinants, can be obtained for example, by well-known reproducible methods from hybridoma-derived antibodies or phage-presenting immunoglobulin libraries. For example, phage display methods can be used to obtain variable domains of human antibodies for an antigen by selecting libraries of human immunoglobulin sequences. The affinity of the initially selected antibodies can be further increased by affinity maturation, for example chain intermixing or random mutagenesis. A person skilled in the art is familiar with methods for obtaining natural or recombinant antibody domains (for laboratory manuals see, for example, Antibody engineering: methods and protocols / edited by Benny K.C. The; Benny K.C. II Series: Methods in molecular biology (Totowa, N.J.)). Generally, any antibody known in the art can be used as a source for the variable domains of the invention.

In a certain aspect of the invention at least one, preferably all of the first VLA domain, the second VHB domain, the third VLB domain and the fourth VHA domain are fully human, humanized or chimeric domains. A humanized variable domain comprises a structural region that substantially has the amino acid sequence of a human immunoglobulin and a CDR of a non-human immunoglobulin. Humanized antibodies can be produced by well-established methods such as, for example, CDR grafting (see for example Antibody engineering: methods and protocols / edited by Benny K.C. Lo, Benny K.C. II Series: Methods in molecular biology (Totowa, N.J.)). Thus, one skilled in the art is readily able to produce a humanized or fully human version of molecules and antigen binding and variable domains from non-human sources, eg murine, with standard molecular biology techniques known in the art for reduce the immunogenicity and improve the efficiency of the antigen binding molecule in a human immune system. In a preferred embodiment of the invention all domains (eg VLA, HBV, VLB and HAV) are humanized or fully human; more preferably, the dimeric antigen binding molecule according to the invention is humanized or fully human. The term "completely human" as used herein means that the amino acid sequences of the variable domains and the peptides that bind the variable domains in the first and second polypeptide chains originate in or may be found in humans. In certain embodiments of the invention, the variable domains can be human or humanized but not the peptides that bind to the variable domains.

In one embodiment the first VLA domain, the second VHB domain, the third VLB domain and the fourth VHA domain are specific for the same antigen, so that the antigen binding sites formed by the domains bind either to the same epitope or different epitopes on the same antigen. In this case the terms "antigen A" and "antigen B" refer to the same antigen. These antigen-binding molecules are monospecific.

In another embodiment the first VLA domain, the second VHB domain, the third VLB domain and the fourth VHA domain are specific for different antigens, so that VLA and VHA form an antigen binding site for an antigen A of a first specificity and HBV and VLB form an antigen binding site for an antigen B of a second specificity. Different antigens can be associated with different cell types or represent different antigens of the same cell type. Those antigen binding molecules according to the invention are bispecific.

In some embodiments, at least one antigen binding site may be specific for a bacterial substance, viral protein, autoimmune marker or an antigen present on a particular cell as a cell surface protein of a B cell, T cell, Killer cell Natural (NK), myeloid cell, phagocytic cell, tumor cell.

In one aspect of the invention the dimeric antigen binding molecule is bispecific and comprises a first specificity for an effector cell and a second specificity for a target cell different from the effector cell. These antigen binding molecules are able to crosslink two cells and can be used as direct effector cells for a specific target. In another aspect of the invention the dimeric antigen binding molecule can be biospecific for a target cell and a molecule selected from the group consisting of a drug, toxin, radionucleotide, enzyme, albumin and lipoprotein, natural ligands such as cytokines or chymosins. If the target molecule is albumin, the albumin or serum albumin can be selected from the group of origins consisting of human, bovine, rabbit, canine and mouse.

"Effector cells" typically refer to cells of the immune system that can stimulate or activate cytotoxicity, phagocytosis, antigen presentation, cytosine release. These effector cells are, for example, but not limited to, T cells, Natural Killer (NK) cells, granulocytes, monocytes, macrophages, dendritic cells, erythrocytes and antigen-presenting cells. Examples of suitable specificities for effector cells include but are not limited to CD2, CD3, CD5, CD28 and other T cell receptor (TCR) components for T cells; CD16, CD38, CD44, CD56, CD69, CD335 (NKp46), CD336 (NKp44), CD337 (NKp30), NKp80, NKG2C and NKG2D for NK cells; CD18, CD64 and CD89 for granulocytes; CD18, CD64, CD89 and mannose receptor for monocytes and macrophages; CD64 and cleft receptor for dendritic cells; CD35 for erythrocytes. In certain aspects of the invention, those specificities, ie cell surface molecules, of the effector cells are suitable for mediating the elimination of cells after the binding of a bispecific antibody as a molecule of the cell surface, and therefore, induce cytolysis or apoptosis.

The "target cells" typically refer to the sites to which the effector cells should be directed to induce or activate the biological response, eg, respective immune. Examples of target cells can be tumor cells or infectious agents such as viral or bacterial pathogens, for example dengue virus, herpes simplex, influenza virus, HIV or cells carrying autoimmune targets such as IL-2, an autoimmune marker or a autoimmune antigen.

In a preferred embodiment of the invention, the dimeric antigen binding molecule is bispecific for a tumor cell and an effector cell, in particular a T cell or an NK cell. Suitable specificities for tumor cells can be tumor antigens and cell surface antigens on the respective tumor cell, for example, specific tumor markers. This bispecific dimer antigen binding molecule binds to the tumor cell and the immune effector cell thereby activating the cytotoxic response induced by the T cell or the NK cell. The term "tumor antigen" as used herein includes the tumor associated antigen (TAA) and the tumor specific antigen (TSA). An "antigen associated with the tumor" (TAA) as used herein, is refers to a protein which is present on tumor cells, and on normal cells during fetal life (antigen once fetal), and after birth in selected organs, but at a much lower concentration than on tumor cells. A ??? it may also be present in the stroma in the vicinity of the tumor cell, but expressed in lower amounts in the stroma anywhere in the body. In contrast, the term "tumor-specific antigen" (TSA) refers to a protein expressed by tumor cells. The term "cell surface antigen" refers to an antigen or fragment thereof that can be recognized by an antibody on the surface of a cell.

Examples of specificity for tumor cells include but are not limited to CD19, CD20, CD30, the laminin receptor precursor protein, EGFR1, EGFR2, EGFR3, Ep-CAM, PLAP, Thomsen-Friedenreich antigen (TF), MUC- 1 (mucin), IGFR, CD5, IL4-R alpha, IL13-R, FceRI and IgE and as described in the art.

In one embodiment, the specificity for an effector cell can be CD3 or CD16 and the specificity for a tumor cell can be selected from CD19, CD20, CD30, the laminin receptor precursor, Ep-CAM, EGFR1, EGFR2, EGFR3, PLAP , Thomsen-Friedenreich antigen (TF), MUC-1 (mucin), IGFR, CD5, IL4-R alpha, IL-13-R, Fc RI and IgE. Particular examples of those antigen binding molecules are bispecific for CD3 and CD19 or CD16 and CD30.

In a certain aspect of the invention, the first VLA domain and the fourth VHA domain have the specificity for a tumor cell and the other two domains, ie, the second HBV domain and the third VLB domain, have the specificity for an effector cell, in particular the T cell or NK cell. In one embodiment, the first VLA domain and the fourth VHA domain have the specificity for one tumor cell and the other two domains, ie the second HBV domain and the third VLB domain, have the specificity for CD3 or CD16. In a certain embodiment thereof the first VLA domain and the fourth VHA domain have a specificity for CD19, CD20, the laminin receptor precursor, Ep-CAM, EGFR1, EGFR2, EGFR3, PLAP, Thomsen-Friedenreich antigen (TF) , MUC-1 (mucin), IGFR, CD5, IL4-R alpha, IL13-R, FcsRI and the other two domains, ie the second HBV domain and the third VLB domain, have a specificity for CD3.

In another aspect of the invention, the first VLA domain and the fourth VHA domain have the specificity for an effector cell, in particular T cell or NK cell, and the other two domains, i.e., the second VHB domain and the third VLB domain they have the specificity for a tumor cell. In one embodiment, the first VLA domain and the fourth VHA domain have the specificity for one CD3 or CD16 and the other two domains, ie the second VHB domain and the third VLB domain, have the specificity for a tumor cell. In a particular preferred embodiment, the first VLA domain and the fourth VHA domain have specificity for one CD3 and the other two domains, ie, the second VHB domain and the third VLB domain have the specificity for a tumor cell selected from the group consisting of of CD19, CD20, CD30, the laminin receptor precursor, Ep-CAM, EGFR1, EGFR2, EGFR3, PLAP, Thomsen-Friedenreich antigen (TF), MUC-1 (mucin), IGFR, CD5, IL4-R alpha , IL13-R, FcsRI and IgE.

The CD3 antigen is associated with the T cell receptor complex on T cells. In the case where the specificity for an effector cell is CD3, the binding of the dimer antigen binding molecule according to the invention to CD3 can activate the cytotoxic activity of T cells on target cells. That is to say, by the biospecific binding of the binding molecule of a dimeric antigen to CD3 and to a white cell, for example, a tumor cell, the cell lysis of the white cell can be induced. Dimeric antigen binding molecules with a specificity towards CD3 and its production are known in the art (and described by example in Kipriyanov et al., 1999, Journal of Molecular Biology 293: 41-56, Le Gall et al., 2004, Protein Engineering, Design & Selection, 17/4: 357-366).

The monospecific anti-CD3 antigen binding molecules are known for their immunosuppressive properties by binding to and modulation of the T cell receptor (for example, as described in WO 2004/024771). In one embodiment, the antigen binding molecule according to the present invention is bispecific for CD3 and albumin to be used as an immunosuppressive agent, for example, in transplantation.

The CD16 antigen (FCYIIIA) is a receptor expressed on the surface of NK cells. The NK cells possess an inherent cytolytic activity and by bispecific binding of the dimeric antigen binding molecule according to the invention to CD16, the cytotoxic activity of the NK cells towards the target cell can be activated. An example of a bispecific antigen binding molecule having specificity for CD16 is described, for example, in Arndt et al., 1999, Blood, 94: 2562-2568). In a particular embodiment of the invention, at least one of the heavy chain and light chain variable domains are of an anti-CD16 antibody described in WO 2006/125668, in particular antibodies that recognize the CD16A isoform, but not the isoform CD16B.

The dimeric antigen-binding molecules according to the invention, where the tumor specificity is towards the CD19 antigen can be used for the immunotherapy of malignant B-cell processes, because the CD19 antigen is expressed on virtually all malignant diseases of lineage B from lymphoblastic leukemia (ALL) to non-Hodgkin's lymphoma (NHL). In particular, for the treatment of non-Hodgkin's lymphoma, the dimeric antigen binding molecules having specificity towards CD19 or CD20 can be used. Dimeric antigen binding molecules that have specificity towards CD19 and its production are known in the art (and are described, for example, in Cochlovius et al., 2000, Cancer Research 60: 4336-4341).

The dimeric antigen binding molecules according to the invention, where the tumor specificity is towards the laminin receptor or the laminin receptor precursor can be used, for example, but not limited, for the treatment of chronic lymphocytic leukemia of B cells (B-CLL), non-Hodgkin's lymphoma, Hodgkin lymphoma, lung cancer, colon carcinoma, breast carcinoma, pancreatic carcinoma, prostate cancer, in particular, in the condition of metastatic cancer or minimal residual cancer. Antigen-binding molecules that have specificity to the receptor precursor of laminin are described, for example, in Zuber et al., 2008, J. Mol. Biol., 378: 530-539.

The dimeric antigen binding molecules according to the invention, where the tumor specificity is towards EGFR1 may be of particular use in the treatment of cancers where the expression of EGFR1 is upregulated or altered, for example, in breast, bladder, head and neck, prostate, kidney, non-small cell lung cancer, colorectal cancer and glioma.

The dimeric antigen binding molecules according to the invention, where the tumor specificity is towards the TF antigen may be particularly useful in the treatment of breast or colon cancer and / or liver metastasis.

Dimeric antigen binding molecules, where the tumor specificity is toward CD30 may be particularly useful in the treatment of Hodgkin's disease. Antigen-binding molecules that have specificity towards CD30 are described, for example, in Arndt et al., 1999, Blood, 94: 2562-2568.

Dimeric antigen binding molecules, where the tumor specificity is towards the alpha chain of the IL-4 receptor (IL4R alpha) may be particularly useful in the treatment of solid tumors, in particular breast carcinomas, ovaries, renal system, head Y neck, malignant melanoma and Kaposi's sarcoma related to AIDS. Dimeric antigen binding molecules, where at least one additional specificity is towards EGFR3 / HER3 and / or EGFR2 / neu may be particularly useful in the treatment of breast cancer. Dimeric antigen binding molecules where tumor specificity is towards IGFR may be particularly useful in the treatment of prostate cancer, colorectal cancer, ovarian cancer or breast cancer.

Dimeric antigen binding molecules, where tumor specificity is toward CD5 may be particularly useful in the treatment of chronic lymphocytic leukemia.

Dimeric antigen binding molecules, where the tumor specificity is towards MUC-I may be particularly useful in the treatment of gastric cancer and ovarian cancer.

Dimeric antigen binding molecules, where the tumor specificity is towards EpCAM may be particularly useful in the treatment of carcinomas of the colon, kidney, and breast.

The dimeric antigen binding molecules, where the tumor specificity is towards PLAP may be of particular use in the treatment of ovarian or testicular cancer.

Dimeric antigen binding molecules, where the tumor specificity is toward OFA-iLR may be particularly useful in the treatment of metastatic tumors.

In certain aspects of the invention, the antigen binding molecule as described herein is dimeric and bispecific for CD3 and CD19 or the antigen binding molecule is dimeric and bispecific for CD18 and CD19. In a particular embodiment thereof, the first VLA domain and the fourth VHA domain are specific for CD3 and CD16, respectively, while the second VHB domain and the third VLB domain are specific for CD19. In both cases, the first and second polypeptide chains each have the domain order VLCD3-VHCD19-VLCD19-VHCD3 or VLCD16-VHCD19-VLCD19-VHCD16 from the N-terminus to the C-terminus of the polypeptide chains. In a preferred embodiment, the first, second, third and fourth domains are humanized or fully human. In a more preferred embodiment the first and second polypeptide chains are defined above are humanized or completely human. In another aspect of the invention, the dimeric antigen binding molecule can be bispecific, for example, for EpCAM and CD3; albumin, such as, for example, HSA and CD3; or EGFR and CD3.

In a further aspect of the invention, the molecule of Antigen binding as described herein is specific for albumin, for example human serum albumin (HSA), and another antigen different from albumin. That antigen binding molecule binds to serum albumin, thereby increasing the serum half-life in vivo. Thus, those antigen binding molecules are advantageous for medical or diagnostic uses and pharmaceutical compositions, wherein the polypeptide of those antigen binding molecules comprises a light chain variable domain and a heavy chain variable domain of a therapeutic antibody. or diagnostic and a light chain variable domain and a heavy chain variable domain specific for albumin. The known and / or commercially available therapeutic, diagnostic or antialbumin antibodies can be used as sources for the variable domains of light chain and heavy chain variable domains. In addition, methods for creating and generating antibodies or Fv fragments specific for albumin, e.g., HSA, are known in the art. In these antigen-binding molecules the domains of the polypeptide chain are arranged in the order VLA-HBV-VLB-HAV, where the antigen A or the B antigen is albumin. In a preferred embodiment the albumin is antigen A. In a certain aspect of the invention, the other antigen is CD3. In a particular embodiment, the A antigen is human serum albumin (HSA) and the polypeptide of a HSAxCD3 antigen-binding molecule has the order VLHSA-VHCD3-VLCD3-VHHSA domain as shown in Example 2. To generate that antigen-binding molecule, for example, the variable domains of antibodies or fragments of anti-HSA antibodies and anti-CD3 can be generated or inserted in the respective order, for example, analogs as described for CD3xCD19 in Example 1 in the expression plasmid shown in Figure 7 replacing the anti-CD3 and anti-CD19 domains shown or any other expression plasmid or suitable expression construct.

A further aspect of the invention provides a dimeric antigen binding molecule according to a previously described embodiment, which is linked to a further functional unit, eg, a domain or functional agent, which mediates a biological function independent, in particular a bio-chemical event. The additional functional unit can be complexed with or covalently linked to at least one of the two individual polypeptide chains of the dimeric antigen binding molecule. In one aspect, the additional functional unit can be covalently bound to only one of the individual polypeptide chains and in another aspect, the additional functional unit can be covalently linked to both polypeptide chains of the dimeric antigen binding molecule thus binding the two polypeptide chains. In one more aspect, each of the two polypeptide chains individually binds covalently to an additional functional unit. When the additional functional unit is covalently linked to at least one of the two polypeptide chains, the additional functional unit can be fused to at least one of the two polypeptide chains by means of a peptide bond or a peptide linker. Alternatively, the additional functional unit can be linked by a chemical conjugation such as a disulfide bridge, for example, between a cysteine residue of at least one polypeptide chain and a cysteine residue of the additional functional unit, ester linkage or by crosslinking chemistry. In a certain aspect of the invention, the additional functional unit can be linked to the antigen binding molecule by a cleavable linker, such as, for example, a disulfide bond.

The additional functional unit can be linked to the N-terminal or C-terminal of the first and / or second polypeptide chains. If an additional functional unit is linked to both the first and second polypeptide chains, the additional functional unit can be linked N-terminal with respect to a polypeptide chain and C-terminal relative to the other polypeptide chain.

Homobifunctional and heterobifunctional reagents for the chemical crosslinking of a polypeptide chain with a further functional unit such as an additional polypeptide or agent are well known in the art. Examples include but are not limited to 5, 5'-dithiobis (2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (o-PDM), succinimidyl 3- (2-pyridyldithio) propionate (SPDP), S-acetylthio N-succinimidyl acetate (SATA), 4- (N-maleimidomethyl) cyclohexan-L-succinimidyl carboxylate (SMCC) or 4- (4-N-maleimidophenyl) butyric acid hydrazide (PBH). Methods for crosslinking polypetide chains comprising immunoglobulin chains with an additional polypeptide or chemical agent are described for example in Graziano et al., Methods in Molecular Blology, 2004, vol. 283, 71-85 and Hermanson, GT "Bioconj ugate Techniques" Academic Press, London 1996.

In one aspect, the additional functional unit may be at least one additional variable immunoglobulin domain. The additional variable immunoglobulin domain may be specific for the first antigen A or the second B antigen for which the binding sites of the dimeric antigen binding molecule are specific or, alternatively, specific for a third C antigen, which is different from antigen A and antigen B. In certain aspect, an additional light chain variable domain VL and an additional heavy chain variable domain VH can be fused to each of the two polypeptide chains, such that an additional domain, in particular VH, is fused at N-terminus and the another additional domain, in particular VL, is fused to the C-terminus resulting in a polypeptide having six variable domains which will be associated with other polypeptides identical to a dimeric antigen binding molecule having six antigen binding sites. In another aspect, an additional variable immunoglobulin domain can be fused to one of the polypeptide chains of the antigen binding molecule which is then non-covalently associated with a complementary variable immunoglobulin domain with the same specificity of a third polypeptide additional, thereby forming an additional antigen binding site between the dimeric antigen binding molecule and the additional third polypeptide. In another aspect, an additional antigen binding unit that includes a scFv or a diabody can be ligated as a functional unit additional to the dimeric antigen binding molecule.

In a certain aspect, the additional functional unit can be at least one additional dimeric antigen binding molecule as described herein. In consecuense, two or more dimeric antigen binding molecules according to the invention can be ligated together to increase the valence and avidity of the antigen binding molecules.

In another aspect, the additional functional unit may be an effector domain that includes the Fe domain, CH2 domain, CH3 domain, hinge domain or a fragment thereof. That unit can confer effector properties on the antigen binding molecule in the case of binding to Fe receptors. These functional units can also be used to increase the serum half-life of the antigen binding molecule.

In another aspect, the additional functional unit can be an enzyme. In the case where the enzyme is capable of converting a prodrug to an active drug, an antigen binding molecule can be used in the antibody-dependent enzyme prodrug therapy (ADEPT). For this, the antigen binding molecule directs the enzyme to the tissue of interest and when the antigen binding molecule binds to the tissue, the prodrug is activated at that site. In addition, the use of bispecific antigen molecules to direct enzymes to produce therapeutic effects on cancer is known in the art, for example, but not limited to bispecific antigen molecules that have CD30 specificities. and alkaline phosphatase which catalyzes the conversion of mitomycin phosphate to mitomycin alcohol, or specificities for placental alkaline phosphatase and β-lactamase which activate anti-cancer prodrugs based on cephalosporin. Suitable are bispecific antigen binding molecules that have specificity for fibrin and tissue plasminogen activator for fibrinolysis and the use of antigen-binding molecules conjugated with enzymes in enzyme-based immunoassays.

In another aspect, the functional unit can be a drug, toxin, radioisotope, lymphokines, chemokines or marker molecule. That antigen binding molecule releases the functional unit to the desired site of action. For example, a chemotherapeutic drug linked to an antigen binding molecule that is specific for a tumor antigen can be released into a tumor cell and the toxins can be released to the pathogens or tumor cells. An antigen binding molecule linked to a toxin can be used to target NK cells or macrophages and are preferably specific for CD16. Examples of a toxin are but are not limited to ribosyl transferase activator, serine protease, calmodulin-dependent adenyl cyclase adenyl cyclase, ribunuclease, DNA alkylating agent or mitosis inhibitor, eg, doxorubicin. The marker molecule can be, for example, a fluorescent molecule, luminescent or radioactively labeled, a metal chelate or an enzyme (for example horseradish peroxidase, alkaline phosphatase, β-galactosidase, malate dehydrogenase, glucose oxidase, urease, catalase, etc.) which, in turn, when it is subsequently exposed to a substrate it will react with the substrate in such a way that it produces a chemical entity which can be detected and can be used for imaging or immunoassays in vivo, when it binds to the binding molecule of antigen according to the invention. When used for an immunoassay, the dimeric antigen binding molecule can also be immobilized on an insoluble support, for example, glass, polystyrene, polypropylene, polyethylene, dextran, nylon, natural and modified celluloses, polyacrylamides, agarose and magnetic beads.

To increase the serum half-life of the antigen-binding molecules according to the invention in the body, the antigen-binding molecule, if desired, can be fused to albumin or pegylated, sialylated or glycosylated (see, for example , Stork et al., 2008, J. Biol. Chem., 283: 7804-7812). Alternatively to an additional albumin fusion to the antigen binding molecule according to the present invention, the antigen binding molecule itself may be specific for the albumin and another antigen as described here above.

The dimeric antigen binding molecule according to any of the embodiments described hereinabove can be produced by expressing polynucleotides encoding the individual polypeptide chains that are associated with each other to form the dimeric antigen binding molecule. Therefore, a further embodiment of the invention are polynucleotides, for example, DNA or RNA, which code for the polypeptide chains of the dimeric antigen binding molecule as described hereinabove.

The polynucleotides can be constructed by methods known to the person skilled in the art, for example, by combining the genes coding for the first VLA domain, the second HBV domain, the third VLB domain and the fourth VHA domain separated by peptide linkers or directly linked by a peptide bond, and optionally a suitable transcription terminator, and expressing these in bacteria or other appropriate expression system. Depending on the vector system and the host used, any number of suitable transcription and translation elements, including inducible constitutive promoters, can be used. The promoter is selected so as to control the expression of the polynucleotide in the respective host cell.

The polynucleotides can be codons optimized with the polarity of the codon being altered to match the particular expression in the chosen host.

The polynucleotide can be inserted into vectors, preferably expression vectors, which represent a further embodiment of the invention. These recombinant vectors can be constructed according to methods well known to the person skilled in the art, see, for example, Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) New York.

A variety of expression / host vector systems can be used to contain and express the polynucleotides encoding the polypeptide chains of the present invention. These include, but are not limited to, microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors, yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors ( for example, baculovirus); plant cell systems transformed with virus expression vectors (eg, cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti plasmids or pBR322), or animal cell systems, for which, for example, viral expression systems can be used.

A particular preferred expression vector for expression in E.coli is psKK (LeGall et al., J. Immunol.Methods (2004) 285 (l): Ill-27) or pcDNA5 (Invitrogen) for expression in mammalian cells. .

Thus, the dimeric antigen binding molecule as described herein can be produced by introducing a polynucleotide or vector encoding the polypeptide chain as described above in a host cell and culturing the host cell under conditions in which the polypeptide chain be expressed. The dimeric antigen binding molecule obtained from the expressed polypeptide chains can be isolated and, optionally, further purified. The conditions for the growth and maintenance of host cells, the expression, isolation and purification of the dimeric antigen binding molecules according to the invention of those host cells are fully described in the art.

In a further embodiment of the invention there are provided compositions comprising a molecule of Dimeric antigen binding or a polypeptide as described hereinabove and at least one additional component. For use in the prevention or treatment of a disease or disorder the composition containing the dimeric antigen binding molecule or the polynucleic acid molecule encoding the polypeptide chains that form the antigen binding molecule is preferably combined with a pharmaceutically carrier acceptable adequate. The term "pharmaceutically acceptable carrier" encompasses in its meaning any support, which does not interfere with the effectiveness of the biological activity of the ingredients and which is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include saline solutions buffered with phosphate, water, emulsions, such as oil / water emulsions, various types of wetting agents, sterile solutions, etc. These supports can be formulated by conventional methods and can be administered to the subject in a suitable dose. Preferably, the compositions are sterile. These compositions may also contain adjuvants such as preservatives, emulsifying agents and dispersing agents. The prevention of the action of microorganisms can be ensured by the inclusion of several antibacterial and antifungal agents. The administration of the appropriate compositions can be effected in different ways, for example, by intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends on the type of therapy and the type of compound contained in the pharmaceutical composition. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, the doses for any patient depend on many factors, including the patient's size, body surface area, age, sex, particular compound to be administered, time and route of administration, type of therapy , general health and other drugs that are being administered concurrently.

The invention further provides a medical use or a method wherein the dimeric antigen binding molecule as described herein above is administered in an effective dose to a subject, for example, a patient, for an immunosuppressive treatment, for example in a transplant, the treatment of an autoimmune disease, inflammatory disease, infectious disease, allergy or cancer (e.g., non-Hodgkin's lymphoma; chronic lymphocytic leukemia; Hodgkin's lymphoma; solid tumors, for example, those that occur in breast cancer; ovarian cancer, colon cancer, kidney cancer, or bile duct cancer; minimal residual disease; metastatic tumors, for example those that metastasize to the lungs, bones, liver or brain). The antigen binding molecule can be used in prophylactic or therapeutic scenarios, alone or in combination with current therapies.

Cancers that can be treated using the antigen-binding molecule of the present invention include but are not limited to primary and metastatic adrenal cortical cancer, anal cancer, aplastic anemia, bile duct cancer, bladder cancer, bone cancer, bone metastasis , CNS tumors, peripheral CNS cancer, breast cancer, Castelman's disease, cervical cancer, childhood non-Hodgkin's lymphoma, cancer of the colon and rectum, endometrial cancer, esophageal cancer, family of Ewin tumors (for example, sarcoma) from Ewin), eye cancer, bladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational profoblastic disease, bile-cell leukemia, Hodgkin's disease, Kaposi's sarcoma, kidney cancer, hypopharyngeal laryngeal cancer, acute lymphocytic leukemia, acute myeloid leukemia, childhood leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, liver cancer ado, lung cancer, tumors lung carcinoids, non-Hodgkin's lymphoma, male breast cancer, malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, proliferative myeloma disorders, cancer of the nasal and paranasal cavity, nasopharyngeal cancer, neuroblastoma, cancer of the oral and oropharyngeal cavity, osteosarcoma, cancer of ovary, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, cancer of the salivary gland, sarcoma (adult soft tissue cancer), skin cancer of the melanoma type, skin cancer of the different type to melanoma, stomach cancer, testicular cancer, thymic cancer, thyroid cancer, uterine cancer (for example uterine sarcoma), vaginal cancer, vulvar cancer and Waldenstrom macrobulinemia.

An "effective dose" refers to amounts of the active ingredient that are sufficient to affect the course and severity of the disease, leading to the reduction or remission of that pathology. An "effective dose" useful in treating and / or preventing those diseases or disorders can be determined using methods known to the skilled person (see for example, Fingi et al., The Pharmacological Basis of Therapeutics, Goddman and Gilman, eds Macmillan Publishing Co ., New York, pp. 1-46 (1975)).

In another aspect of the invention, the dimeric antigen binding molecule as described herein previously it is used in the preparation of an immunosuppressant medicament or medicament for the treatment of an autoimmune disease, inflammatory disease, infectious disease, allergy or cancer (for example Hodgkin's lymphoma, chronic lymphoma leukemia, Hodgkin's lymphoma, solid tumors, for example those that occur in breast cancer, ovarian cancer, kidney cancer, or cancer of the bile duct, minimal residual disease, metastatic tumors for example those that metastasize the lungs, bones, liver or brain). Where multispecific binding molecules, previously specified as having particular utility in the treatment of a specific disease, have been described, those binding molecules can also be used in the preparation of a medicament for that specified disease.

The methods for preparing pharmaceutical compositions, ie medicament, and the clinical application of the antigen-binding molecules in the prevention and / or treatment of diseases such as, for example, cancer, are known to the person skilled in the art.

In a particular aspect of the invention the dimeric antigen binding molecule is bispecific and used for cancer therapy, because those antibodies can be used to redirect the cells cytotoxic effectors against tumor cells. This therapeutic concept is well known in the art. For example, clinical studies showed tumor regression in patients treated with anti-CD3 x bispecific antitumor antibody (for example Canevari, S. et al., J. Nati. Cancer Inst., 87: 1463-1469, 1996) or treated patients with anti-CD16 x bispecific antitumor antibody (for example Hartman et al., Clin Cancer Res. 2001; 7 (7): 1873-81) The proof of concept has also been shown for several recombinant bispecific antibody molecules comprising only domains variables (Fv) such as, for example, dimeric and tetravalent CD3xCD19 antigen binding molecules having a domain order VHA-VLB-HBV-VLA (Cochlovius et al., Cancer Research, 2000, 60: 4336-4341) or recently in clinical studies with monomeric single chain Fv antibody molecules of the BiTE® format (two single chain antibodies of different specificities linked together; Micromet AG, Germany; Bargou R. et al., Science, 2008, 321 (5891) : 974-977; Baeuerle PA and Reinhardt C, Cancer Res. 2009, 69 (12): 4941-944). The dimeric antigen binding molecules described herein can be used as medicaments and applied in treatment methods in a manner similar to the bispecific antibodies of the art, since they are capable of redirecting the mechanisms Therapeutics, for example cytotoxic ones, using the same combined antibody specificities. In addition, monospecific immunosuppressive antibodies for CD3 such as Muromo-CD3 are known for the treatment of transplant rejection, acute rejection of renal transplants (allografts), liver and heart transplants. Thus, the antigen-binding molecules specific for albumin and CD3 can be used in the same treatment methods as monospecific anti-CD3 antibodies. In addition, the antigen-binding molecules specific for albumin and another antigen, i.e. the therapeutic or diagnostic target, as described herein, can be used for the respective clinical applications of antigen specificity different from that of albumin.

The antigen binding molecule and compositions thereof may be in oral, intravenous, intraperitoneal or other pharmaceutically acceptable dosage forms. In some embodiments, the composition is administered orally and the dosage form is a tablet, capsule, caplet (tablet in the form of a capsule) or another orally available form. In some embodiments, in the parenteral compositions for example intravenous, intraperitoneal, intramuscular or subcutaneous and is administered by means of a solution containing the antigen binding molecule.

One skilled in the art will readily be able to build and obtain the antigen binding molecules disclosed herein using standard techniques and methods known in the art, see for example Sambrook, Molecular cloning A Laboratory Manual, Cold Spring Harbor Laboratory (1989) NY; The Protein Protocols Handbook, edited by John M. Walker, Humana Press Inc. (2002); o Antibody engineering: methods and protocols / edited by Benny K.C. The; Benny K.C. II Series: Methods in molecular biology (Totowa, N.J.)). Furthermore, as one skilled in the art, it will be possible to produce the antigen binding molecules described herein using standard methods known in the art and by modifying the methods described in US 7, 129, 330 Kipriyanov et al. J. Mol. Biol. (1999) 293, 41-56 or Le Gall et al., 2004, Protein Engineering 17: 357-366 so that dimeric antigen binding molecules are obtained as described above comprising two polypeptide chains having the order of VLA-VHB-VLB-VHA domain from N-terminal to C-terminal of each polypetidic chain.

The following examples best illustrate the invention without limiting the scope of the invention.

Example 1: To build functional dimeric tandem diabodies (TandAb®) using a domain array other than VHA-VLB-VHB-VLA, several dimeric tandem diabodies were constructed with the VLA-VHB-VLB-VHA domain array according to the invention using the two domains of a humanized anti-CD19 single chain antibody and a single chain anti-CD3 humanized antibody, respectively. The findings were confirmed using two variants of each antigen binding molecule, which represent the products of different stages of an affinity mutation procedure that was carried out for both humanized anti-CDl9 and humanized anti-CD3 antibodies.

The murine monoclonal antibodies HD37 and UCHT directed against CD19 and CD3, respectively, were the initial material to obtain humanized antibodies with relatively high affinities. In each case the VH domain was first combined with a human VL library in a phagemid scFv vector to select a suitable human VL chain by phage display. In a second step the selected human VL chain was combined with a library of VH domains in which the CDR3 region remained constant. This procedure resulted in a humanized anti CD19 and anti CD3, respectively, which only contained a short murine sequence in the VHCDR3 region. These clones were subsequently matured by affinity introducing point mutations in residues that were thought to be involved in the union of the antigen. The best binding mutants were then selected by phage display. The clones chosen to construct TandAb were M13 and M39 that bind CD19 and C4 and LcHC21 that binds CD3.

The following antibodies were generated: Antibody Al: CD19M39xCD3c4 (option 0) VHCD3C4- TT CD19M39_ CD19 39_TT CD3C4 * L * H "L Antibody B: CD19M39xCD3c4 (option 2) Vlcd3c4-T, CD19M39 TT CD19M39 TT CD3C4 H "VL -VH Antibody A2: CD19M13xCD3LCHC21 (option 0) "CD3LCHC21 t CD19M13 TT CD19 13 TT CD3LCHC21 H "VL ~ H" VL Antibody C: CD19M13xCD3LCHC21 (option 2) vLCD3LCHC21-and CD19M13 _ CD19M13 _ and CD3LCHC21 * H * L "H The plasmids encoding the hybrid monomers vLCD3c4-VHCD19M39-VLCDl9M39-VHCD3c4 of antibody B and VLCD3LCHC21_VHCDI9MI3_VLCDI9MI3_VHCD3LCHC21 q antibody c were generated by a DNA design and processing provider. The backbone of the monomer sequence vLCD3C4_ VHCD19M39_VLCDI9M39_VHCD3C4 comprises the DNA sequences of two scFv antibodies, ie scFvCD19M39 and scFvCD3c4, respectively. The monomeric sequence vLCD3LCHC21-VHCD19M13-VLCDI9MI3_VHCD3LCHC21 combines the variable domains of Fv CD19M13 from a single strand and Fv CD3LCHC21 from a single strand. The four scFv were obtained by presentation selection of phage of single-chain antibodies against CD19 and CD3 antigens. In both cases the sequence information was used to build the previous hybrid monomers. The 9 amino acid (G2S) 3 linker was used to link the domains together. The synthesized gene encoding VLCD3C4-VHCD19M39-VLCD19M39-Vhcd3C4 was cloned into the mammalian expression vector pCDNA5FRT (Invitrogen). The VLCD3LCHC21-VHCD19M13-Vlcd19m13 -Vhcd3lchc21 gene was also cloned into an expression vector and amplified by PCR using a forward primer introducing a Ncol cleavage site of a reverse primer introducing a NotI cleavage site. After analysis and isolation by agarose gel, the PCR product was subsequently digested twice by Ncol and Notl and cloned into the vector psKK3 linearized with Ncol and Notl. The correct cloning was confirmed by DNA sequencing.

The vector map of pCDNA5FRT codes for antibody B shown in Figure 6.

The vector map of pSKK3 encoding antibody C is shown in Figure 7.

For high-level production, the vector containing the gene VLCD3C4_VHCDI9M39_VLCDI9M39_VHCD3C4 was transfected transiently (using Ca3 (P04) 2 in adherent HEK293 cells Fermentation of the protein was carried out under well-known growth conditions in the technique .

The recombinant protein was expressed as a His-Tag fusion protein with a signal peptide. The protein was isolated from the cell culture supernatant by immobilized metal affinity chromatography (IMAC) as described (Kipriyanov et al., 1999, J. Mol. Biol., 293, 41-56). The purified material was subsequently analyzed by SDS-PAGE. Coomassie staining of the SDS-PAGE gel and size exclusion chromatography on a calibrated Super-dex 200 HR10 / 30 column (Amersham Pharmacia, Freiburg, Germany) in sodium phosphate buffer (30 mM Na3P04, 0.75 M arginine / HC1, pH 6.0) revealed a pure recombinant protein assembled correctly (antibodies B).

For high level expression, the gene coding for the used monomer VLCD3LCHC21-VHCD19M13-VLCD19M13-VH CD3LCHC21 followed by a 6x His-Tag was cloned into the plasmid pSKK3 containing the cell suicide system of the hok / sok gene and a Skp gene coding for the periplasmic factor Skp / OmpH (LeGall et al., 2004, J. Immunol.

Methods, 285, 111-127). The plasmid was transfected into a strain of E. coli K12 (ATCC 31608MR).

Transformed bacteria were grown in shake flasks and induced essentially as described above (Cochlovius et al., 2000, J.

Immunol. , 165, 888-895). The recombinant proteins were isolated from the soluble periplasmic fraction and the supernatant of the bacterial medium by immobilized metal affinity chromatography (IMAC) as already described (Kipriyanov et al., 1999, J. Mol. Biol., 293, 41-56 ).

The purified material was further analyzed by SDS-PAGE stained with Coomassie blue and size exclusion chromatography on a calibrated Superdex 200 HR10 / 30 column (Amersham Pharmacia, Freiburg, Germany) in sodium phosphate buffer (30 mM Na3P04, 0.75 M of arginine / HCl, pH 6.0). The product appeared to be pure and correctly assembled.

The comparative antibodies Al and A2 were generated in the same way as antibodies B and C, respectively, where the domain order of antibodies Al and A2, respectively, was reversed compared to that of antibodies B and C, respectively.

The cytotoxicity tests were carried out essentially according to that described by T. Dreier et al. (2002, Int. J. Cancer 100, 690-697). The PMBCs that were used as effector cells were isolated from the peripheral blood of healthy volunteers by density gradient centrifugation. In some cases, PBMC were grown overnight in the presence of 25 U / mL of IL-2 before they were used as effector cells in the cytotoxicity assay. The purity and expression of the antigen of the isolated PBMC was verified by flow cytometry in each case (data not shown).

CD19 + JOK-1 or Raji cells were cultured in RPMI 1640 medium supplemented with 10% FCS, 2 mM L-glutamine and 100 IU / mL of sodium penicillin G and 100 g / mL of streptomycin sulfate (here referred to as RPMI medium). all components of Invitrogen). For the cytotoxicity assay the cells were labeled with calcein AM 10 μ? (Molecular Probes / Invitrogen) for 30 min in RPMI medium without FCS at 37 ° C. After gently washing, the labeled cells were resuspended in RPMI medium at a density of lxl05 / mL. Then lxlO4 white cells were seeded together with 5xl05 PBMC with the indicated antibodies in individual wells of a 96 well microplate with rounded bottom in a total volume of 200 μL / well. After centrifugation for 2 min at 200 g the assay was incubated for 4 hours at 37 ° C in a humidified atmosphere with 5% C02. 15 min before the end of the incubation, 20μL of 10% Triton X-100 in RPMI medium was added to the wells with only target cells. To all the other wells, 20 pL of RPMI medium were added. 100 L of cell culture supernatant was harvested per well after centrifugation additional for 5 min at 500 g, and the fluorescence of the released calcein was measured at 520 nm using a plate fluorescence reader (Victor 3, Perkin Elmer). On the basis of the measured counts, the specific cell lysis was calculated according to the following formula: [fluorescence (sample) - fluorescence (spontaneous)] / [fluorescence (maximum) - fluorescence (spontaneous)] x 100%. The fluorescence (spontaneous) represents the fluorescent counts of target cells in the absence of effector cells and antibodies and the (maximum) fluorescence represents the total cell lysis induced by the addition of Triton X-100. The sigmoidal dose response curves and EC50 values were calculated using the Prism software (GraphPad Software).

Results: The results of cytotoxicity assays for tandem diabodies that have the following order of domain starting at the N-terminus of VHA-VLB-HBV-VLA (antibody A) and VLA-HBV-VLB-VHA (antibody B), respectively , using the M39 variant of the anti CD3 and the C4 variant of the anti CD3 are shown in Figure 3.

Surprisingly, there was a very large difference in the cytotoxic activity of the two tandem diabodies. The tandem diabody having the domain arrangement according to the invention designated as "B antibody" was more than 60x more active than the tandem diabody designated "antibody B" as determined by a comparison of its EC50 values under the given conditions.

The superiority of the domain arrangement represented by the present invention (antibody C) for better cytotoxicity was confirmed using two additional variants of the anti CD19 and anti CD3 antibodies (see Figure 4).

The EC50 value of the tandem diabody with the domain order according to the invention represented by option 2 is extremely low (0.1 pM). This is 27x more active than the TandAb represented by option 0 after comparing the CE5o values under the given conditions.

Example 2: Modulation of the cellular receptor by antibodies (HSA) xCD3 TandAb of human serum albumin in vitro To determine whether antibodies HSAxCD3 TandAb with different domain orders differ in their efficacy to induce modulation of the T cell receptor (TCR) / CD3 on T cells in vitro Jurkat CD3 + cells were cultured in the presence of increasing concentrations of antibodies HSAxCD3 TandAb bispecific and subsequently analyzed by the remaining TCR. Essay Modulation was carried out in the presence or absence of HSA to measure the influence of HSA on the activity of TandAbs.

Briefly, Jurkat cells were seeded in individual wells of a 96-well microplate with a rounded bottom in RPMI 1640 medium supplemented with 2mM L-glutamine and 100 IU / mL of sodium penicillin G and 100 pg / mL of sulfate. streptomycin (all components of Invitrogen). In a separate microplate, Jurkat cells were seeded in RPMI medium as described above but with the addition of 50 mg / ml HSA (Sigma). After the addition of the indicated antibodies, the cells were incubated in a total volume of 200 L / well at 37 ° C in a humidified incubator in the presence of 5% C02. As a control, the cells were cultured in the absence of antibodies. After washing with ice-cold phosphate-buffered saline (PBS, Invitrogen, Karlsruhe, Germany) supplemented with 2% heat-inactivated FCS (Invitrogen, Karlsruhe, Germany) and 0.1% sodium azide (Roth, Karlsruhe, Germany) ) (referred to as FACS buffer) the cells were stained with 10 μ? of anti-TCR antibody to / β conjugated to PC5 (Beckman-Coulter) in a total volume of 100 iL in FACS buffer for 45 on ice in the dark. After washing twice with shock absorber FACS was measured by fluorescence of 10 cells at 675 nm with a FC500 MPL flow cytometer (Beckman-Coulter). The mean fluorescence values were determined using the CXP software (Beckman-Coulter) and used for the 4-parameter nonlinear / logistic regression analysis using the GraphPad Prisme version 3.03 for Windows, GraphPad Software, San Diego, California, USA .

The results obtained from the TCR CAB-306 modulation experiment described in figure 5 and summarized in Table 1 demonstrate the comparable TCR modulation efficiency of both HSAxCD3 TanAb in the order of domain VHA-VLB-VHB-VLA, ( option 0) and VLA-HBV-VLB-VHA (option 2) which demonstrates the effectiveness of the modulation when the HSA is the B antigen. However, in the presence of physiological concentrations of HSA the modulation efficiency in the case of the TandAb (VHA-VLB-VHB-VLA) of option 0 decreased considerably, while the value of EC50 for TandAb in orientation (VLA-VHB-VLB-VHA) of option 2 was increased only by a factor of 2.6 .

These data clearly indicate the superior properties of the HSAxCD3 TanAb in the VLA-VHB-VLB-VHA domain orientation of option 2 when compared to the HSAxCD3 TandAb (VHA-VLB-VHB-VLA) of option 0.

Table 1: Summary of the results of the TCR modulation experiment: the EC50 values of the TCR modulation experiment with the two antibodies HSAxCD3 TandAb in the presence or absence of HSA (Figure 5, experiment CAB-306) were determined by adjusting 4-parameter non-linear / logistic regression.

Although the preferred embodiments of the present invention have been shown and described here, it will be obvious to those skilled in the art that those embodiments are provided by way of example only. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed to practice the invention. It is intended following claims define the scope of the invention and that the methods and structures within the scope of those claims and their equivalents are covered by them.

Claims

1. A dimeric antigen binding molecule consisting of a first and a second polypeptide chain, each of the first and second polypeptide chains contains - a first VLA domain that is a light chain variable domain specific for an antigen A; - a second a VHB domain that is a heavy chain variable domain specific for a second B antigen; - a third VLB domain that is a light chain variable domain specific for the second antigen A; and - a fourth a HAV domain which is a heavy chain variable domain specific for the first antigen A; characterized in that - the domains arrange in each of the first and second of the polypeptide chains in the order VLA-VHB-VLB-VHA from the N-terminal to the C-terminal of the polypeptide chains and - the first VLA domain of the first polypeptide chain is associated with the fourth VHA domain of the second polypeptide chain to form an antigen binding site for the first antigen A; - the second HBV domain of the first polypeptide chain is associated with a third VLB domain of the second polypeptide chain to form an antigen binding site for the second antigen B; -the third VLB domain of the first polypeptide chain is associates with the second HBV domain of the second polypeptide chain to form an antigen binding site for the first antigen B; and - the fourth VHA domain of the first polypeptide chain is associated with the first VLA domain of the second polypeptide chain to form an antigen binding site for the first antigen A.

2. The antigen binding molecule according to claim 1, characterized in that the first and second polypeptide chains are not covalently associated.

3. The antigen binding molecule according to claim 1 or 2, characterized in that the antigen binding molecule is tetravalent.

4. The antigen binding molecule according to any of claims 1 to 3, characterized in that the antigen binding molecule is bispecific and the first antigen A or the second antigen B is albumin.

5. The antigen binding molecule according to claim 4, characterized in that the antigen binding molecule is bispecific for (i) HSA and CD3, (ii) CD3 and CD19 or (iii) CD16 and CD19.

6. The antigen binding molecule according to any of claims 1 to 5, characterized in that the domains are human or humanized domains.

7. The antigen binding molecule according to any of claims 1 to 6, characterized in that the antigen binding molecule comprises at least one additional functional unit.

8. The antigen binding molecule according to any of claims 1 to 7, characterized in that the antigen binding molecule is specific for a B cell, T cell, natural killer (NK) cell, myeloid cell or phagocytic cell.

9. The antigen binding molecule according to any of claim 8, characterized in that the antigen binding molecule is bispecific, antigen binding molecule which is specific for a tumor cell.

10. The antigen binding molecule according to any of claim 9, characterized in that the first light chain variable domain (VLA) and the first heavy chain variable domain (HAV) are specific for a tumor cell.

11. A suitable polypeptide chain for forming a dimeric antigen binding molecule according to any of claims 1 to 10 with another second polypeptide chain consisting of - a first VLA domain that is a light chain variable domain specific for a first antigen A, - one second a HBV domain that is a heavy chain variable domain specific for a second B antigen, - a third VLB domain that is a light chain variable domain specific for the second B antigen; and - a fourth a VHA domain which is a heavy chain variable domain specific for the first A antigen, characterized in that the domains are arranged in the polypeptide chain in the order VLA-VHB-VLB-VHA from N-terminal to C-terminal of the polypeptide chains, where the second VHB domain and the third VLB domain are covalently connected by a short central linker L2, so that a single chain Fv antigen binding unit for the B antigen is formed by the two domains Adjacent VHB and VLB is avoided.

12. The polypeptide chain according to claim 11, characterized in that the domains are specific for (i) CD3 and CD19 (ii) CD16 and CD19 or (iii) albumin and CD3.

13. A nucleic acid molecule, characterized in that it encodes a polypeptide chain according to claim 11 or 12.

14. A composition, characterized in that it comprises the antigen-binding molecule according to any of claims 1 to 10, the polypeptide chain according to claim 11 or 12 or the nucleic acid molecule according to claim 13 and a pharmaceutically acceptable carrier.

15. The antigen binding molecule according to any of claims 1 to 10 for use as a medicament.