WO2015036582A2 - Tetravalent homodimeric antigen-binding proteins - Google Patents

Abstract

The present invention relates to homodimeric tetravalent antigen-binding polypeptides, either monospecific or bispecific, polynucleotides encoding said binding polypeptides, expression vectors comprising these polynucleotides and prokaryotic or eukaryotic host cells comprising these polynucleotides or vectors. Furthermore, the invention relates to therapeutic and diagnostic uses of said structures in the fields of oncology, inflammatory and immune disorders.

Description

TETRAVALENT HOMODIMERIC ANTIGEN-BINDING PROTEINS Technical Field

The present invention relates to Domain-Rearranged Engineered Antibody Molecules (“DREAM”), and uses thereof in the treatment of a variety of diseases and disorders, including cancer and immunological and inflammatory disorders. The domain-rearranged antibody molecules of the invention are symmetric; they comprise at least two identical polypeptide chains that associate to form at least four antigen-binding sites, which may recognize the same or different epitopes. Additionally, the epitopes may be from the same or different antigens located on the same or different cells. The individual polypeptide chains of the DREAMs may be covalently linked through the covalent bonds, such as, but not limited, disulphide bonding of cysteine residues located within each polypeptide chain. In particular embodiments, the tetravalent homodimeric molecules of the present invention further comprise the constant domain of the antibody light chain (C-kappa or C-lambda), which allows stabilization of the multimeric antibody constructs.

Background Art

The recent clinical and commercial success of therapeutic antibodies has generated great interest in antibody-based therapeutics for haematological malignancies, solid tumours, autoimmune and inflammatory diseases (Rothe et al., 2008, "Therapeutic advances in rheumatology with the use of recombinant proteins", Nat Clin Pract Rheumatol 4:605-14; Argyriou and Kalofonos, 2009, "Recent advances relating to the clinical application of naked monoclonal antibodies in solid tumors", Mol Med 15:183-91; Chan and Carter, 2010, "Therapeutic antibodies for autoimmunity and inflammation", Nat Rev Immunol 10:301-16).

Being highly specific, naturally evolved molecules, the antibodies are able to bind to primary and metastatic cancer cells with high affinity and cause the destruction of tumour cells by complement-dependent cytotoxicity (CDC), by antibody-dependent cellular cytotoxicity (ADCC), and/or by delivering an apoptotic signal to a target cell. Although therapeutic monoclonal antibodies have become a major, often well-tolerated treatment modality for many cancer patients, their efficacy needs further improvement. Malfunction of naked immunoglobulins in some therapeutic settings is accounted for by FcγRIIIa (CD16a) polymorphism (Cartron, 2009, "FCGR3A polymorphism story: a new piece of the puzzle", Leuk Lymphoma 50:1401-2), interaction of antitumor antibodies with inhibitory Fc receptors (e.g., FcγRIIb) on myeloid cells (Clynes et al., 2000, "Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets", Nat Med 6:443-6) and by different escape mechanisms developed by cancer cells to evade mortality (Baeuerle et al., 2003, "Bispecific antibodies for polyclonal T-cell engagement", Curr Opin Mol Ther 5:413-9).

To generate more potent antibodies that work better in combination or possibly as monotherapy, different enhancement approaches have been designed (Beck et al., 2010, "Strategies and challenges for the next generation of therapeutic antibodies", Nat Rev Immunol 10:345-52). One alternative immunotherapeutic strategies is based on the activation of host immune mechanisms using bispecific antibodies (Kipriyanov and Le Gall, 2004, "Recent advances in the generation of bispecific antibodies for tumor immunotherapy", Curr Opin Drug Discov Devel 7:233-42; Kiprijanov, 2011, "Bispecific Antibodies and Immune Therapy Targeting", Drug Delivery in Oncology: From Basic Research to Cancer Therapy 2:441-82).

Bispecific antibodies are man-made proteins which are able binding two targets simultaneously. They can override the natural specificity of an immunological effector cell for its target and redirect lysis towards a cell population it would otherwise ignore. Designed to direct and enhance the body’s immune response to specific tumours, these bispecific antibodies consist of a targeting domain (typically an antibody fragment that binds to, e.g., a tumour antigen) linked to a triggering arm that is specific for another antigen which could be a cell-surface molecule capable of mediating a phagocytic or lytic response by immune effector cells, or a growth factor, or even a toxic payload. This property enables developing therapeutic strategies that are not possible with conventional monoclonal antibodies.

Recent clinical success of the trioma-made anti-human EpCAM / anti-human CD3 half-mouse/half-rat bispecific antibody catumaxomab (Removab^®) followed by its approval in Europe confirmed the therapeutic potential of bispecific antibodies (Bokemeyer, 2010, "Catumaxomab--trifunctional anti-EpCAM antibody used to treat malignant ascites", Expert Opin Biol Ther 10:1259-69). However, a major limitation of the bispecific antibodies produced by hybrid hybridomas (quadromas) (Milstein and Cuello, 1983, "Hybrid hybridomas and their use in immunohistochemistry", Nature 305:537-40) or by using a trioma (cross-species hybridoma) technology (Mocikat et al., 1997, "Trioma-based vaccination against B-cell lymphoma confers long-lasting tumor immunity", Cancer Res 57:2346-9) is their immunogenicity. Repeated doses of rodent antibodies elicit an anti-immunoglobulin antibody response, which compromises therapy with bispecific antibody. For example, roughly one third of the patients treated with the trioma-made antibodies develop immune reaction to mouse or rat protein (HAMA/HARA response) (Kiewe and Thiel, 2008, "Ertumaxomab: a trifunctional antibody for breast cancer treatment", Expert Opin Investig Drugs 17:1553-8).

An intact unmodified antibody of IgG class is a heterotetramer comprising two heavy and two light polypeptide chains. The N-terminal parts of the heavy and light chain, the so-called variable (V) domains (V_H and V_L, respectively), form the antigen-binding ‘fragment variable’ (Fv) of an antibody. The domain architecture of antibodies and the advances in recombinant DNA technology provide an opportunity to develop methods for engineering and producing bispecific antibodies exclusively from the antigen-binding (Fv) antibody fragments (Kipriyanov and Le Gall, 2004, "Recent advances in the generation of bispecific antibodies for tumor immunotherapy", Curr Opin Drug Discov Devel 7:233-42; Chames and Baty, 2009, "Bispecific antibodies for cancer therapy: the light at the end of the tunnel?", MAbs 1:539-47).

To stabilize the Fv modules, a peptide linker was introduced between the variable domains of the antibody heavy and light chain with the formation of the so-called single-chain (sc) Fv molecules (Huston et al., 1988, "Protein engineering of antibody binding sites: recovery of specific activity in an anti-digoxin single-chain Fv analogue produced in Escherichia coli", Proc Natl Acad Sci U S A 85:5879-83).

Two scFv-based bispecific antibody formats have been intensively studied, tandem scFvs or (scFv)₂ (Mack et al., 1995, "A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity", Proc Natl Acad Sci U S A 92:7021-5) and diabodies (Holliger et al., 1993, ""Diabodies": small bivalent and bispecific antibody fragments", Proc Natl Acad Sci U S A 90:6444-8; Johnson et al., 2010, "Effector cell recruitment with novel Fv-based dual-affinity re-targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion", J Mol Biol 399:436-49). In a first approach, the individual protein domains, such as heavy and light chain antibody variable domains (V_H and V_L, respectively) from two antibodies of different specificity, are fused together as a single polypeptide chain in an order, e.g., V_L ^A-V_H ^A-V_H ^B-V_L ^B (where A and B indicate different specificities), and the functional antigen-binding Fv modules are formed from the adjacent complementary domains separated by peptide linkers of more than 12 amino acids (aa). This format has been used for generation of bispecific T-cell engager (BiTE^®) antibodies which showed high potency in killing tumour cells by T-cell recruitment both in vitro (Löffler et al., 2000, "A recombinant bispecific single-chain antibody, CD19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated T lymphocytes", Blood 95:2098-103; Dreier et al., 2002, "Extremely potent, rapid and costimulation-independent cytotoxic T-cell response against lymphoma cells catalyzed by a single-chain bispecific antibody", Int J Cancer 100:690-7; Löffler et al., 2003, "Efficient elimination of chronic lymphocytic leukaemia B cells by autologous T cells with a bispecific anti-CD19/anti-CD3 single-chain antibody construct", Leukemia 17:900-9) and in animal models (Dreier et al., 2003, "T cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemic human B cell lymphoma xenografts by a CD19-/CD3- bispecific single-chain antibody construct", J Immunol 170:4397-402), and demonstrated promising results in clinical trials (Bargou et al., 2008, "Tumor regression in cancer patients by very low doses of a T cell-engaging antibody", Science 321:974-7; Nagorsen et al., 2009, "Immunotherapy of lymphoma and leukemia with T-cell engaging BiTE antibody blinatumomab", Leuk Lymphoma 50:886-91; Topp et al., 2009, "Report of a Phase II Trial of Single-Agent BiTE ^(R) Antibody Blinatumomab in Patients with Minimal Residual Disease (MRD) Positive B-Precursor Acute Lymphoblastic Leukemia (ALL)", Blood (ASH Annual Meeting Abstracts) 114:840). This format is also disclosed in EP 107 17 52 and WO 99/54440.

In the second method, the recombinant bispecific molecules are formed by non-covalent association of two hybrid scFvs, e.g., such as V_H ^A-V_L ^B and V_H ^B-V_L ^A, each comprising V_H and V_L domains of different specificity (A and B, respectively), separated by a short peptide linker (<12 amino acids) that prevents intramolecular V_H/V_L pairing, thus giving a four domain bispecific diabody (Kipriyanov et al., 1998, "Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells", Int J Cancer 77:763-72). In general, diabodies are well folded molecules and, unlike (scFv)₂, can be easily produced with high yields in bacteria (Zhu et al., 1996, "High level secretion of a humanized bispecific diabody from Escherichia coli", Biotechnology (N Y) 14:192-6; Cochlovius et al., 2000, "Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells", J Immunol 165:888-95). They have also demonstrated high activity in recruitment of either T cells or NK cells to kill tumour cells both in vitro and in animal models (Kipriyanov et al., 1998, "Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells", Int J Cancer 77:763-72; Arndt et al., 1999, "A bispecific diabody that mediates natural killer cell cytotoxicity against xenotransplanted human Hodgkin's tumors", Blood 94:2562-8; Cochlovius et al., 2000, "Treatment of human B cell lymphoma xenografts with a CD3 x CD19 diabody and T cells", J Immunol 165:888-95; Kipriyanov et al., 2002, "Synergistic antitumor effect of bispecific CD19 x CD3 and CD19 x CD16 diabodies in a preclinical model of non-Hodgkin's lymphoma", J Immunol 169:137-44). However, co-secretion of two hybrid scFv fragments forming a bispecific diabody can give rise to two types of dimer: active heterodimers and inactive homodimers, thus decreasing the proportion of the functional bispecific product. Therefore, the mismatch of non-complementary V_H and V_L domains is a major issue in manufacturing bispecific diabodies.

Unlike native antibodies, which are themselves dimeric and thus bivalent, there is only one binding domain for each specificity in both mentioned above scFv-scFv tandem and the bispecific diabody formats. Bivalent binding is an important means of increasing the functional affinity, and possibly the selectivity, of antibodies and antibody fragments for particular cell types carrying densely clustered antigens. In addition, small size of both scFv-scFv tandems and diabodies (50-60 kDa) leads to their rapid clearance from the blood stream through the kidneys, thus making the drug administration process less convenient. For example, the BiTE^® antibody blinatumomab was administered in clinical trials by continuous infusion over 4-8 weeks in order to maintain adequate serum exposure (Bargou et al., 2008, "Tumor regression in cancer patients by very low doses of a T cell-engaging antibody", Science 321:974-7).

A multimeric Fv antibody consisting of four antibody variable domains arranged in an orientation V_H ^A-L1-V_L ^B-L2-V_H ^B-L3-V_L ^A (where “A” and “B” are different specificities) has been described (Kipriyanov et al., 1999, "Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics", J Mol Biol 293:41-56). The four V_H and V_L domains are bound into a single-chain construct by three peptide linkers, L1, L2 and L3. The linkers L1 and L3 between the V-domains of different specificity are short (less than 12 amino acid residues) and do not allow formation of Fv modules from the neighbouring V_H and V_L domains. The middle linker L2 is either short (12 amino acid residues or less), which leads to formation of a tandem diabody (all antigen-binding sites are formed by the respective V_H or V_L domains of two different polypeptide chains with four domains each), or the linker L2 is rather long (27 amino acid residues), which leads to the formation of a single-chain diabody (all antigen-binding sites are formed by the respective V_H and V_L domains of the same polypeptide chain). The same formats are disclosed in EP1078004 and US7129330. However, the single-chain diabody of the prior art is also relatively small (50-60 kDa) and has the same disadvantages, as the above-mentioned scFv-scFv tandems and diabodies. Although the tetravalent bispecific tandem diabody is larger and can bind both antigens bivalently, it is stabilized only by the non-covalent association of two V_H/V_L pairs and, therefore, is relatively unstable and has relatively high aggregation propensity.

Thus, the technical problem underlying the present invention is to provide the new multimeric antigen-binding structures that overcome disadvantages of the Fv-antibodies of the prior art and to provide a general way to form a stable polypeptide molecule with at least four antigen-binding sites, which is monospecific or multispecific.

The solution of said technical problem is achieved by providing the embodiments characterized in the claims.

Disclosure of Invention

The present invention relates to the symmetric multimeric antigen-binding polypeptides and to their use in the treatment of a variety of diseases and disorders including cancer, autoimmune disorders, allergy, inflammatory disorders and infectious diseases caused by the viruses, bacteria or fungi. Preferably, the multimeric antigen-binding structures of the present invention can bind to at least two different epitopes on two different cells wherein the first epitope is expressed on a different cell type than the second epitope, such that the multimeric molecule can cross-link the two cells together.

The present invention is based on the complementarity of the cognate V_H and V_L domains derived from the same antibody and their ability to form heterodimers. Although in the most cases stability of a single Fv module (non-covalent V_H/V_L heterodimer) is low, with a dissociation constant (K _D) in the range of 1-10 µM (Glockshuber et al., 1990, "A comparison of strategies to stabilize immunoglobulin Fv-fragments", Biochemistry 29:1362-7), the single-chain polypeptides comprising several V_H and V_L domains can form relatively stable homo- and heteromeric complexes due to an avidity effect. This was demonstrated for scFv molecules (comprise one V_H and one V_L domain) which under certain conditions can form dimers (diabody), trimers (triabody), tetramers (tetrabody), and even higher multimeric forms (Holliger et al., 1993, ""Diabodies": small bivalent and bispecific antibody fragments", Proc Natl Acad Sci U S A 90:6444-8; Le Gall et al., 1999, "Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding", FEBS Lett 453:164-8; Todorovska et al., 2001, "Design and application of diabodies, triabodies and tetrabodies for cancer targeting", J Immunol Methods 248:47-66). Accordingly, a four-domain single-chain diabody (comprises two V_H and two V_L domains) can form even a more stable homodimer, so-called tandem diabody (TandAb^®), where all four V-domains are involved into interchain pairing (Kipriyanov et al., 1999, "Bispecific tandem diabody for tumor therapy with improved antigen binding and pharmacokinetics", J Mol Biol 293:41-56; Cochlovius et al., 2000, "Cure of Burkitt's lymphoma in severe combined immunodeficiency mice by T cells, tetravalent CD3 x CD19 tandem diabody, and CD28 costimulation", Cancer Res 60:4336-41). However, both diabodies and the TandAbs^® mentioned above are the non-covalent dimers formed in anti-parallel (head-to-tail) orientation. The present invention provides a general way of making a more stable covalently linked antibody-like multimeric molecule with at least four antigen-binding sites, which is monospecific or bispecific. Each monomer of the single-chain molecule of the present invention comprises five antibody-derived protein domains; four of them are variable domains (two pairs of V_H and V_L domains) and a single light chain constant domain (C_L), C-kappa or C-lambda, in the middle of the molecule. The presence of the C_L domain, which is able to form C_L/C_L homodimers, provides an opportunity of parallel (head-to-head) dimerization of the whole single-chain polypeptide and of covalent stabilization of the generated protein complex due to formation of the interchain disulphide bond by the unpaired cysteines located at the C-terminal part of the C_L domain. In addition, the single-chain approach used in the present invention eliminates the need to co-express two different polypeptide chains and, therefore, reduces the product heterogeneity.

The multimeric mono- or bispecific antigen-binding structures of the present invention are expected to be very stable and have a higher antigen-binding capacity. In addition, having a molecular weight of approximately 125-150 kDa, which is far above the renal threshold, they should have favourable pharmacokinetics making them particularly useful for therapeutic purposes, since the monomeric scFv-scFv tandems and dimeric diabodies of the prior art are fairly small (approx. 50 kDa) and are quickly eliminated from the blood stream through kidney filtration. Moreover, the single-chain format and lack of glycosylation of the multimeric antibodies of the present invention allows them to be produced in different expression systems, such as bacteria, yeast, plants, insect cells and mammalian cells.

The present invention relates to a symmetric homodimeric structure formed by a single-chain protein molecule that comprises four antibody variable domains and a single antibody light chain constant domain, C-kappa or C-lambda, wherein the individual domains are separated by the peptide linkers of different length and

1. the first or last two variable domains bind intermolecularly with the complementary V_H or V_L domains of another protein chain to form the antigen-binding V_H/V_L pairs;
2. the single C_L domain is located in the middle of the polypeptide chain (between two sets of the variable domains) and is involved in association with the identical C_L domain of another protein chain with the formation of the interchain disulphide bond in the C_L/C_L interface to stabilize the dimeric protein complex;
3. the other two variable domains bind either each other intramolecularly within the same polypeptide chain with the formation of an antigen-binding scFv module or interact with the complementary V_H and V_L domains of another protein chain to form the functional antigen-binding V_H/V_L pairs.

In a particularly preferred embodiment, the present invention relates to a multimeric antibody-like molecule characterized by the following features:

1. the monomers of said Fv-antibody comprise at least four antibody variable domains (V_H and V_L) and a single constant domain of the antibody light chain (C_L);
2. the adjacent V_H and V_L domains are derived from the same antibody and are separated by the peptide linkers either of more than 12 amino acids to form functional Fv modules within the same polypeptide chain or of less than 12 amino acids to facilitate dimerization of the single-chain polypeptide.

A further preferred feature is that the antigen-binding V_H and V_L pairs (in V_H-to-V_L or in V_L-to-V_H orientation) of the same specificity are linked to either the N-terminus of C_L via a linker of less than 5 amino acids or to the C-terminus of the C_L domain through a peptide linker of 0-20 amino acid residues.

The term “peptide linker” relates to any peptide capable of connecting two antibody domains with its length depending on the kinds of domains to be connected. The peptide linker may contain any amino acid residue with the amino acids glycine (Gly) and serine (Ser) being preferred.

The term “intramolecularly” means interaction between the V_H and V_L domains belonging to the same polypeptide chain (monomer) with the formation of functional antigen-binding site.

The term “intermolecularly” means interaction of the cognate V_H and V_L domains, which belong to different monomers.

The term “valency” refers to the number of potential antigen-binding sites in a polypeptide. A polypeptide may be monovalent and contain one antigen-binding site or a polypeptide may be bivalent and contain two antigen-binding sites. Additionally, a polypeptide may be tetravalent and contain four antigen-binding sites. Each antigen-binding site specifically binds one antigen. When a polypeptide comprises more than one antigen-binding site, each antigen-binding site may specifically bind the same or different antigens. Thus, a polypeptide may contain a plurality of antigen-binding sites and, therefore, be multivalent and a polypeptide may specifically bind the same or different antigens.

The term “specificity” refers to the number of potential antigen-binding sites, which immunoreact with (specifically bind) a given antigen, in a polypeptide. The polypeptide may be a single polypeptide or may be two or more polypeptides joined by non-covalent interactions or by disulphide bonding. A polypeptide may be monospecific and contain one or more antigen-binding sites specifically interacting with the same antigen or a polypeptide mat be bispecific and contain two or more antigen-binding sites, which are able specifically bind two immunologically distinct antigens. Thus, a polypeptide may contain a plurality of antigen-binding sites, which specifically bind the same or different antigens.

The term “multimeric” refers to a polypeptide comprising more than one polypeptide. A multimer may be dimeric and contain two polypeptides and a multimer may be trimeric and contain three polypeptides. Multimers may be homomeric and contain two or more identical polypeptides or a multimer may be heteromeric and contain two or more non-identical polypeptides.

The dimeric or multimeric antigen-binding constructs of the present invention can be prepared according to the standard methods and protocols. Preferably, the gene coding for the monomeric polypeptide chain is prepared by ligation of the DNA sequences encoding the peptide linkers with the genes of the antibody variable (V_H and V_L) or constant (C-kappa or C-lambda) domains. The genes of the antibody domains are generated either by chemical synthesis or are produced by a polymerase chain reaction (PCR) from a complementary DNA (cDNA) derived from messenger RNA (mRNA) isolated either from the hybridoma cells or from other source of antibody genes (e.g., isolated immune B cells, peripheral blood lymphocytes, spleens and/or tonsils). The assembled gene encoding the monomer of the antibody-like multimeric molecule is ligated into a suitable expression vector for generation of the recombinant protein in the corresponding host cells.

The antigen-binding structures of the present invention can comprise at least one further protein domain linked by the covalent or non-covalent bonds. The linkage can be based on genetic fusion according to the methods known in the art or can be performed by, e.g., chemical cross-linking. The additional domain carrying, e.g., toxic payload (Pseudomonas or Shiga toxin, etc.) or detection/purification tag (e.g., His₆ tag) may preferably be linked by a flexible linker, preferably peptide linker, wherein said peptide linker comprises hydrophilic amino acid residues and is of length sufficient to span the distance between the C-terminus of the said further protein domain and the N-terminus of the antigen-binding structure of the present invention or vice versa. The above described fusion protein may further comprise a cleavable linker or a cleavage site for the proteinases.

In a preferred embodiment of the present invention, the antigen-binding molecules are monospecific. The order of domains in a monomer and the linkers separating them may give rise to the following structures (see also Figures 1-3):

1-1 Db ^A -C _L /C _L -scFv ₂ ^A (Figure 1)

1-1a V_H ^A-L1-V_L ^A-C_L-L2-V_L ^A-L3-V_H ^A (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

1-1b V_H ^A-L1-V_L ^A-C_L-L2-V_H ^A-L3-V_L ^A (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

1-1c V_L ^A-L1-V_H ^A-C_L-L2-V_L ^A-L3-V_H ^A (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

1-1d V_L ^A-L1-V_H ^A-C_L-L2-V_H ^A-L3-V_L ^A (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

1-2 scFv ₂ ^A -C _L /C _L -Db ^A (Figure 2)

1-2a V_H ^A-L1-V_L ^A-C_L-L2-V_L ^A-L3-V_H ^A (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-2b V_H ^A-L1-V_L ^A-C_L-L2-V_H ^A-L3-V_L ^A (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-2c V_L ^A-L1-V_H ^A-C_L-L2-V_L ^A-L3-V_H ^A (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-2d V_L ^A-L1-V_H ^A-C_L-L2-V_H ^A-L3-V_L ^A (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-3 Db ^A -C _L /C _L -Db ^A (Figure 3)

1-3a V_H ^A-L1-V_L ^A-C_L-L2-V_L ^A-L3-V_H ^A (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-3b V_H ^A-L1-V_L ^A-C_L-L2-V_H ^A-L3-V_L ^A (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-3c V_L ^A-L1-V_H ^A-C_L-L2-V_L ^A-L3-V_H ^A (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

1-3d V_L ^A-L1-V_H ^A-C_L-L2-V_H ^A-L3-V_L ^A (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

wherein “Db” is a diabody module formed by interchain pairing of the cognate V_H and V_L domains; “scFv” is an scFv module formed by intrachain pairing of the adjacent V_H and V_L domains; L1, L2 and L3 are the peptide linkers connecting the individual antibody domains into a single-chain polypeptide; “A” is an antibody specificity.

In a further preferred embodiment of the present invention, the antigen-binding molecules are bispecific. The order of domains in a monomer and the linkers separating them may give rise to the following structures (see also Figures 4-6):

2-1 Db ^A -C _L /C _L -scFv ₂ ^B (Figure 4)

2-1a V_H ^A-L1-V_L ^A-C_L-L2-V_L ^B-L3-V_H ^B (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

2-1b V_H ^A-L1-V_L ^A-C_L-L2-V_H ^B-L3-V_L ^B (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

2-1c V_L ^A-L1-V_H ^A-C_L-L2-V_L ^B-L3-V_H ^B (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

2-1d V_L ^A-L1-V_H ^A-C_L-L2-V_H ^B-L3-V_L ^B (L1 < 12 aa; L2 = 0-20 aa; L3 > 12 aa)

2-2 scFv ₂ ^A -C _L /C _L -Db ^B (Figure 5)

2-2a V_H ^A-L1-V_L ^A-C_L-L2-V_L ^B-L3-V_H ^B (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-2b V_H ^A-L1-V_L ^A-C_L-L2-V_H ^B-L3-V_L ^B (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-2c V_L ^A-L1-V_H ^A-C_L-L2-V_L ^B-L3-V_H ^B (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-2d V_L ^A-L1-V_H ^A-C_L-L2-V_H ^B-L3-V_L ^B (L1 > 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-3 Db ^A -C _L /C _L -Db ^B (Figure 6)

2-3b V_H ^A-L1-V_L ^A-C_L-L2-V_H ^B-L3-V_L ^B (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-3a V_H ^A-L1-V_L ^A-C_L-L2-V_L ^B-L3-V_H ^B (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-3c V_L ^A-L1-V_H ^A-C_L-L2-V_L ^B-L3-V_H ^B (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

2-3d V_L ^A-L1-V_H ^A-C_L-L2-V_H ^B-L3-V_L ^B (L1 < 12 aa; L2 = 0-20 aa; L3 < 12 aa)

wherein “Db” is a diabody module formed by interchain pairing of the cognate V_H and V_L domains; “scFv” is an scFv module formed by intrachain pairing of the adjacent V_H and V_L domains; L1, L2 and L3 are the peptide linkers connecting the individual antibody domains into a single-chain polypeptide; “A” and “B” are different antibody specificities.

In some cases, it might be desirable to strengthen the association of the two variable domains. Accordingly, in a further preferred embodiment of the present invention, binding of at least one V_H/V_L pair is strengthened by at least one interdomain disulphide bond. This can be achieved by modifying the DNA sequences encoding the variable domains by introducing the codons for the amino acid cysteine. The two most promising sites for introducing disulphide bridges appear to be V_H ⁴⁴-V_L ¹⁰⁰ connecting a framework-2 of the heavy chain with a framework-4 of the light chain, and V_H ¹⁰⁵-V_L ⁴³ that links the V_H framework-4 with the V_L framework-2.

For the particular therapeutic applications, at least one monomer of the present invention can be covalently or non-covalently linked to a biologically active protein (e.g., cytokine, chemokine or growth factor), a chemotherapeutic agent (e.g., doxorubicin, cyclosporine, etc.), an anti-neoplastic agent (e.g., monomethyl auristatin, calicheamicins, etc.), peptide (e.g., alpha-amanitin), a protein toxin (e.g., Pseudomonas exotoxin, ricin, etc.), a protease (e.g., granzyme A and B), or radioactively labelled.

In a preferred embodiment, the multimeric antibody-like construct of the present invention is a monospecific antibody capable of specifically binding to a G-protein coupled receptor (GPCR), preferably a chemokine receptor (e.g., CCR4, CCR5, CXCR3, CXCR4, etc.), or a tumour-associated antigen (such as Axl, CD19, CD20, CEA, EGFR, EpCAM, FGFR, HER2, HER3, etc.), or a tumour-promoting growth factor (e.g., VEGF, angiopoietin-2, etc.), or a chemokine (e.g., CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, etc.).

In a further preferred embodiment, the multimeric antibody-like construct of the present invention is a monospecific biparatopic antibody capable of specific binding to the different epitopes on the same antigen from the group of GPCR, preferably the chemokine receptor (e.g., CCR4, CCR5, CXCR3, CXCR4, etc.), or tumour-associated antigens (such as Axl, CD19, CD20, CEA, EGFR, EpCAM, FGFR, HER2, HER3, etc.), or tumour-promoting growth factors (e.g., VEGF, angiopoietin-2, etc.), or the chemokines (e.g., CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1, etc.).

In an even more preferred embodiment, the multimeric antibody-like construct of the present invention is a bispecific antibody capable of specific binding to the following antigen pairs present on the same or different cells:

• Axl × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• Axl × EGFR (HER1);
• Axl × HER2;
• Axl × HER3;
• CD19 × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• CD20 × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• CD19 × CD20;
• CD19 × CD22;
• CD20 × CD22;
• CD20 × CXCR4;
• CEA × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• CEA × EpCAM;
• EGFR (HER1) × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• EGFR (HER1) × CEA;
• EGFR (HER1) × EpCAM;
• EGFR (HER1) × HER2;
• EGFR (HER1) × HER3;
• EpCAM × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• EpCAM × CCR4;
• EpCAM × CXCR4;
• HER2 × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• HER2 × CEA;
• HER2 × CXCR4 (or CCR4, or CCR7, or S₁P₁);
• HER2 × EpCAM;
• HER2 × HER3;
• HER3 × CEA;
• HER3 × EpCAM.

In a further preferred embodiment, the multimeric antibody-like construct of the present invention is a bispecific antibody capable of specific binding to the cell-surface antigen (such as Axl, CCR4, CXCR4, CEA, EpCAM, HER1, HER2, HER3, etc.) and to the soluble serum protein (e.g., VEGF, angiopoietin-2, human serum albumin, etc.).

Another object of the present invention is a process for the preparation of a multimeric antigen-binding structure, wherein the gene coding for the monomeric polypeptide chain is prepared by ligation of the DNA sequences encoding the peptide linkers with the genes of the antibody variable (V_H and V_L) or constant (C-kappa or C-lambda) domains. The genes of the antibody domains are generated either by chemical synthesis or are amplified by PCR from cDNA derived of mRNA isolated either from the hybridoma cells or from other source of the antibody genes (e.g., isolated immune B cells, peripheral blood lymphocytes, spleens, tonsils). The assembled gene encoding the monomer of the antibody-like multimeric molecule is ligated into a suitable expression vector for generation of the recombinant protein in the corresponding host cells.

The present invention also relates to the DNA sequences encoding the multimeric antigen-binding structures of the present invention and to the vectors, preferably expression vectors containing said DNA sequences.

A variety of the expression vectors and host systems may be utilized for propagation and expression of the DNA sequences encoding the multimeric antibody structures. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage, plasmid, phagemid, or cosmid DNA expression vectors; yeast (Saccharomyces, Pichia or other) transformed with yeast expression vectors; insect cells transformed with the corresponding plasmid-like expression vectors or infected with the baculovirus expression vectors; plant systems transformed with the plasmid or viral expression vectors; avian cells, such as DT40, EB66, etc., and mammalian cells, such as Chinese Hamster Ovary (CHO), human embryonic kidney cells (HEK-293), PER.C6, etc., stably or transiently transformed with the corresponding expression vectors.

The present invention also relates to a pharmaceutical composition containing a multimeric antigen-binding polypeptide of the present invention, a DNA sequence or an expression vector, preferably combined with the suitable pharmaceutical carriers known in the art. Such carriers can be formulated by conventional methods and can be administered to the subject at a suitable dose. Administration of the suitable compositions may be performed by different ways, e.g. by single injections or by continuous infusion using different administration routes, such as intravenous (IV), intraperitoneal (IP), subcutaneous (SC), intramuscular (IM), intravitreal (IVT), intradermal (ID) route. Alternatively, the suitable composition may be administered via a non-invasive route, such as topical (e.g., as eye drops), intranasal or pulmonary (e.g., in a form of spray).

Preferred medical uses of the compounds of the present invention are: (a) treatment of cancer (haematological, solid, metastatic, minimal residual disease); (b) treatment of inflammatory and immune disorders (such as rheumatoid arthritis, systemic lupus erythematosus, inflammatory bowel disease, allergic asthma, idiopathic pulmonary fibrosis, etc.); (c) treatment of infectious diseases caused by the viruses, bacteria, fungi or which are prion-related.

A further object of the present invention is the use of a multimeric antigen binding structure for the diagnostic purposes. The corresponding diagnostic tests are provided by the present invention, such as the kits comprising a multimeric antibody or a combination of several multimeric antibodies of the present invention. The compound of the present invention can be detectably labelled with a radioisotope or fluorophore. In a preferred embodiment, said diagnostic test is used in a form of known in the art enzyme-linked immunosorbent assay (ELISA), Gyrolab^® immunoassay platform or medical imaging.

The present invention is further described with regard to the Figures.

The following examples illustrate the invention.

EXAMPLE 1: Construction of the plasmids for expression of the multimeric antigen-binding molecules in bacteria and mammalian cells

For generation of CD19 × CD3 bispecific molecules, previously described variable domains of the antibodies specific for the human B-cell antigen CD19 and the human T-cell antigen CD3 have been used. For all cloning steps and DNA isolation, the Escherichia coli K12 strain XL1-Blue^® (Stratagene, La Jolla, CA) was used. The plasmids pHOG21-HD37 (Kipriyanov et al., 1996, "Rapid detection of recombinant antibody fragments directed against cell-surface antigens by flow cytometry", J Immunol Methods 196:51-62) and pHOG21-dmOKT3 (Kipriyanov et al., 1997, "Two amino acid mutations in an anti-human CD3 single chain Fv antibody fragment that affect the yield on bacterial secretion but not the affinity", Protein Eng 10:445-53) encoding the scFv fragments specific either for human CD19 or for human CD3, respectively, were used as a source of genetic information for assembly of the genes for the mono- and bispecific antibodies. Generation and characterization of monospecific anti-CD19 and anti-CD3 scFv species with different linkers between the variable domains were performed essentially as described previously (Le Gall et al., 1999, "Di-, tri- and tetrameric single chain Fv antibody fragments against human CD19: effect of valency on cell binding", FEBS Lett 453:164-8; Le Gall et al., 2004, "Immunosuppressive properties of anti-CD3 single-chain Fv and diabody", J Immunol Methods 285:111-27). These genes were combined in tandems using the DNA blocks encoding the linkers L1, L2 and L3 (Figure 7). For expression in bacteria, the assembled genes coding for either monospecific or bispecific molecules followed by His₆ tail were transferred as the NcoI/XbaI fragments into a plasmid pSKK3 containing the hok/sok plasmid-free cell suicide system and an skp gene encoding the Skp/OmpH periplasmic factor (Le Gall et al., 2004, "Immunosuppressive properties of anti-CD3 single-chain Fv and diabody", J Immunol Methods 285:111-27).

For expression in mammalian cells, such as human embryonic kidney cells (HEK-293) or Chinese hamster ovary cells (CHO), the mammalian expression vectors, such as pLNO (Norderhaug et al., 1997, "Versatile vectors for transient and stable expression of recombinant antibody molecules in mammalian cells", J Immunol Methods 204:77-87), were used. The sequences of all newly constructed genes and plasmids were verified by restriction digests and sequencing.

EXAMPLE 2: Expression and purification of recombinant proteins

The E. coli K12 strain RV308 (

lac

74 galISII::OP308strA) (Maurer et al., 1980, "Gene regulation at the right operator (O _R ) bacteriophage l. I. O _R 3 and autogenous negative control by repressor", J. Mol. Biol. 139:147-61) (ATCC 31608) was used for functional expression of single-chain antibodies. The bacteria transformed with the expression plasmids were grown in shaking flasks and induced essentially as described previously (Kipriyanov, 2009, "High-level periplasmic expression and purification of scFvs", Methods Mol Biol 562:205-14). The recombinant proteins were isolated from soluble periplasmic fractions by immobilized metal affinity chromatography (IMAC) followed by ion-exchange chromatography as described previously (Kipriyanov, 2009, "Generation of bispecific and tandem diabodies", Methods Mol Biol 562:177-93). Protein concentrations were determined by the Bradford dye-binding assay (Bradford, 1976, "A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding", Anal. Biochem. 72:248-54) using the Bio-Rad protein assay kit (Bio-Rad Laboratories AB, Oslo, Norway). The purified material was subjected to analytical size-exclusion chromatography on a calibrated Superdex^® 200 10/300 GL column (GE Healthcare, Uppsala, Sweden) in PBSI buffer (15 mM sodium phosphate, 0.15 M NaCl, 50 mM imidazole, pH 7.0).

For the mammalian cell expression, the corresponding plasmids were transfected into HEK-293T cells with FuGENE transfection reagent (Promega, Madison, WI). The transiently transfected cells were propagated in Nunc EasyFill Cell Factories (Thermo Fischer Scientific) and the protein molecules were purified to homogeneity from the cell culture supernatants by affinity chromatography on HiTrap^® Protein L column (GE Healthcare) followed by ion-exchange chromatography on SP-Sepharose^® and gel filtration on HiLoad 16/60 Superdex^® 200 column (GE Healthcare) equilibrated in PBS, pH 7.4.

The recombinant product isolated either from the bacterial periplasmic fractions or from the supernatants of mammalian cells appeared to be a homogeneous dimeric protein with a molecular mass of approximately 150 kDa. When electrophoresed under non-reducing conditions, the molecule migrates as a disulphide-stabilized dimer having a molecular weight of approximately 150 kDa. When electrophoresed under reducing conditions, the molecule migrates as a monomer having a molecular weight of approximately 75 kDa.

EXAMP LE 3: Flow cytometry and cell-surface retention

The cell binding activity of generated multimeric molecules was analysed by flow cytometry. The human CD3⁺/CD19^- acute T cell leukaemia line Jurkat (ATCC TIB-152) and the CD19⁺/CD3^- Burkitt’s lymphoma cell line Raji (ATCC CCL-86) were used for flow cytometry as described previously (Kipriyanov et al., 1998, "Bispecific CD3 x CD19 diabody for T cell-mediated lysis of malignant human B cells", Int J Cancer 77:763-72). In brief, the cell lines were cultured under the conditions recommended by the supplier. For flow cytometry, a total of 10⁵ cells were incubated with 100 µL PBS (Life Technologies) supplemented with 0.2 % BSA and 0.09% sodium azide (Roth, Karlsruhe, Germany) (referred to as FACS buffer) and containing diluted recombinant antibodies for 1 hr on ice at 4 ºC. After washing with FACS buffer, the cells were stained with 1 μg/mL of R-Phycoerythrin (RPE)-conjugated goat anti-human IgG (AbD Serotec, Düsseldorf, Germany) for 30 min at 4 ºC. The stained cells were washed and resuspended in 200 µL of FACS buffer containing 2 µg/mL propidium iodide (Sigma-Aldrich) to exclude dead cells. The fluorescence of stained cells was measured using either an EasyCyte flow cytometer (Guava Technologies, Hayward, CA) or a FACSCanto II flow cytometer (BD Biosciences, San Jose, CA). Median fluorescence intensity values were plotted against the antibody concentration and the experimental data were analysed using a ‘one site – total binding’ equation of the software program PRISM version 5.04 (GraphPad Software, San Diego, CA).

In order to investigate the biological relevance of the molecular form compositions in different preparations of bispecific molecules, the in vitro retention of the multimeric protein structures on the surface of both CD19⁺ and CD3⁺ cells was determined by flow cytometry. In contrast to the direct binding experiments, the cell surface retention and deduced affinity constants (off-rate) are not dependent on the accuracy of determining the concentration of the biologically active protein. The cell surface retention assays were performed at 37 °C under conditions preventing internalization of cell surface antigens as described previously (Adams et al., 1998, "Increased affinity leads to improved selective tumor delivery of single-chain Fv antibodies", Cancer Res 58:485-90), except that the detection of retained antibody was performed using anti-(His)₆ mouse MAb 13/45/31-2 (10 µg/mL; Dianova) followed by FITC-conjugated goat anti-mouse IgG (15 µg/mL; Dianova). The kinetic dissociation constant (k _off) and t _1/2 values for dissociation of protein molecules were deduced from a one-phase exponential decay fit of experimental data using the software program PRISM (GraphPad Software, San Diego, CA).

Cell binding and cell-surface retention experiments demonstrated that the isolated homodimeric molecule is bispecific and tetravalent. It could bind both CD19⁺ and CD3⁺ cells with avidity similar to that of the parental bivalent anti-CD19 and anti-CD3 antibodies, respectively.

EXAMPLE 4: Cellular cytotoxicity mediated by the bispecific molecules

The CD19⁺ Raji target cells, cultivated under standard conditions, were collected by centrifugation, washed twice and re-suspended in RPMI-1640 medium. One mL containing 2.5 × 10⁶ cells was mixed with calcein-AM (Life Technologies) to a final concentration of 10 µM and incubated at 37 °C for 30 min. The cells were washed three times in RPMI-1640/ 10% FBS and the cell density was adjusted to 3 × 10⁵ cells/mL. Human peripheral blood mononuclear cells (PBMC) were prepared from fresh donor blood by Ficoll-Hypaque gradient centrifugation, washed in RPMI-1640/ 10% FBS and re-suspended at a density 6 × 10⁶ cells/mL. Fifty µL of the target and effector cells were mixed in the same wells of a 96-well microtiter plate thus providing an effector-to-target (E:T) cell ratio of 20:1. The CD19 × CD3 bispecific molecules were added to the same wells and the plate was incubated at 37 °C for four hrs. After 3 hrs and 45 min incubation, 20 µL 0.9% Triton X-100 was added to the control wells to achieve complete lysis of the target cells (referred as maximal lysis). One hundred µL supernatant of each sample was then transferred into a black microtiter plate and the fluorescence (excitation at 488 nm, emission at 518 nm) was recorded using a Tecan M200 plate reader. Each experiment was carried out in quadruplicate. The fluorescence intensity of the samples without bispecific molecules was subtracted as a background and the percentage of specific lysis in samples with antibodies was calculated. To determine EC ₅₀ values (effective concentrations leading to 50% maximal killing), the dose-response curves were computed by a nonlinear regression analysis and a three-parameter fit model ‘log [agonist] vs. response’ using the software program PRISM (GraphPad).

The results demonstrated high cytotoxic activity of the homodimeric bispecific molecule of the present invention with EC ₅₀ values below 0.1 ng/mL.

EXAMPLE 5: Testing biological activity in an animal model

The anti-tumour activity of tetravalent bispecific CD19 × CD3 molecules was analysed in a mouse model of human B-cell lymphoma. Immunodeficient NOD/SCID mice (Taconic, Denmark) were used in the experiment. The mouse strain NOD/SCID combines the severe combined immune deficiency mutation (SCID) with the Non-obese diabetic (NOD) background. As a result, these mice lack mature T cells, B cells, and display a reduced NK cell activity due to NOD background (Taconic Immunology Brochure). In order to confirm the immunodeficient status, the animals were tested for IgG production by determination of serum IgG using commercial mouse IgG ELISA kit (Roche Applied Science). The mice were maintained under sterile and standardized environmental conditions (20 ± 1 °C room temperature, 50 ± 10% relative humidity, 12-h light-dark rhythm) and received autoclaved food and bedding and acidified (pH 4.0) drinking water ad libitum. For the model establishment, CD19⁺ Raji cells (human Burkitt’s lymphoma; 2.5 × 10⁶ cells) were premixed with 10⁷ freshly isolated human PBMC and inoculated subcutaneously (SC) in a total volume 0.2 mL / mouse. Intravenous treatment with bispecific antibodies (at doses 50 µg or 0.5 mg per kg body weight) or the vehicle (PBS) started two hours after cell inoculation and was repeated on a daily basis at five consecutive days. The tumour sizes were measured twice a week with a calliper in two perpendicular dimensions. Tumour volumes were calculated according to (width² × length) × 0.5 as a correlate for efficacy. Body weight of mice was determined twice per week as the indicator for tolerability of treatment. The tumour growth curves for the non-treated and different treatment groups were compared and the statistical significance of tumour volume data was calculated by two-way ANOVA with Bonferroni post-test using software PRISM (GraphPad).

At both tested dosages, the dimeric tetravalent molecule of the present invention demonstrated high anti-tumour activity. At lowest dose (50 µg/kg), statistically significant retardation of the tumour growth was observed. At the highest dose (0.5 mg/kg), all treated mice remained tumour-free until end of the experiment (6 weeks), while all vehicle-treated animals reached the non-tolerated tumour sizes within 4 weeks.

Brief Description of Drawings

FIGURE 1: Schematic representation of the domain organization in a single-chain monomeric precursor and a putative structure of a folded dimeric tetravalent monospecific antigen-binding molecule of the present invention in orientation Db^A-C_L/C_L-scFv₂ ^A. “A” is an antibody specificity.

FIGURE 2: Schematic representation of the domain organization in a single-chain monomeric precursor and a putative structure of a folded dimeric tetravalent monospecific antigen-binding molecule of the present invention in orientation scFv₂ ^A-C_L/C_L-Db^A. “A” is an antibody specificity.

FIGURE 3: Schematic representation of the domain organization in a single-chain monomeric precursor and a putative structure of a folded dimeric tetravalent monospecific antigen-binding molecule of the present invention in orientation Db^A-C_L/C_L-Db^A. “A” is an antibody specificity.

FIGURE 4: Schematic representation of the domain organization in a single-chain monomeric precursor and a putative structure of a folded dimeric tetravalent bispecific antigen-binding molecule of the present invention in orientation Db^A-C_L/C_L-scFv₂ ^B. “A” and “B” are different antibody epitope specificities.

FIGURE 5: Schematic representation of the domain organization in a single-chain monomeric precursor and a putative structure of a folded dimeric tetravalent bispecific antigen-binding molecule of the present invention in orientation scFv₂ ^A-C_L/C_L-Db^B. “A” and “B” are different antibody epitope specificities.

FIGURE 6: Schematic representation of the domain organization in a single-chain monomeric precursor and a putative structure of a folded dimeric tetravalent bispecific antigen-binding molecule of the present invention in orientation Db^A-C_L/C_L-Db^B. “A” and “B” are different antibody epitope specificities.

FIGURE 7: Amino acid sequence of the single-chain protein monomer corresponding to the tetravalent bispecific CD19 × CD3 molecule in orientation Db^CD19-C_L/C_L-scFv₂ ^CD3.

Claims (28)

1. A tetravalent homodimeric antigen-binding protein comprising two identical polypeptide chains where each monomer comprises at least four antibody variable domains and a single antibody light chain constant domain, wherein (a) either the N-terminal or the C-terminal two variable domains bind intermolecularly with the complementary V_H or V_L domains of another protein chain to form the antigen-binding V_H/V_L pairs, and (b) the other two variable domains bind either each other intramolecularly within the same polypeptide chain with the formation of an antigen-binding scFv module or interact with the complementary V_H and V_L domains of another protein chain to form the functional antigen-binding V_H/V_L pairs, and (c) the single antibody light chain constant domain is located between the two sets of variable domains disclosed in parts (a) and (b) and is involved in association with the identical antibody light chain constant domain of another protein chain with the formation of the dimeric protein complex.
2. The tetravalent homodimeric antigen-binding protein of claim 1, wherein the adjacent V_H and V_L domains are derived from the same antibody and are separated by the peptide linkers of either more than 12 amino acids to form functional Fv modules within the same polypeptide chain or less than 12 amino acids to facilitate dimerization of the single-chain polypeptide.
3. The tetravalent homodimeric antigen-binding protein of claim 1 or 2, wherein the antibody light chain constant domain is C-kappa or C-lambda.
4. The tetravalent homodimeric antigen-binding protein of claim 1 to 3, which has at least four antigen-binding sites.
5. The tetravalent homodimeric antigen-binding protein of claim 1 to 4, wherein the antigen-binding V_H and V_L pairs (in V_H-to-V_L or in V_L-to-V_H orientation) of the same specificity are linked to either the N-terminus of the antibody light chain constant domain via a peptide linker of less than 5 amino acids or to the C-terminus of the antibody constant domain through a peptide linker of 0-20 amino acid residues.
6. The tetravalent homodimeric antigen-binding protein of claim 1 to 5, wherein the homodimeric structure is stabilized by at least one interchain disulphide bond.
7. The tetravalent homodimeric antigen-binding protein of claim 6, wherein the disulphide bond is formed between the antibody constant domains.
8. The tetravalent homodimeric antigen-binding protein of claim 6, wherein the disulphide bond is located in the interface formed by the antibody variable domains.
9. The tetravalent homodimeric antigen-binding protein of claim 1 to 8, which is monospecific.
10. The tetravalent homodimeric antigen-binding protein of claim 1 to 8, which is bispecific.
11. The tetravalent homodimeric antigen-binding protein of any claim 1 to 10, wherein the monomer is covalently or non-covalently linked to a biologically active protein, a chemotherapeutic agent, an anti-neoplastic agent, a peptide, a protease, or radioactively labelled.
12. The tetravalent homodimeric antigen-binding protein of any claim 1 to 11, which is capable of specific binding to a G-protein coupled receptor.
13. The tetravalent homodimeric antigen-binding protein of claim 12, where the G-protein coupled receptor is a chemokine receptor, such as but not limited to CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or HCMV encoded chemokine receptor.
14. The tetravalent homodimeric antigen-binding protein of claim 12, where the G-protein coupled receptor is angiotensin II receptor AT₁, beta-adrenergic receptor, bradykinin receptor, cannabinoid receptor, cholecystokinin A receptor, endothelin 1 receptor, free fatty acid receptor, Frizzled, gastric-inhibitory-peptide-receptor, gastrin-releasing peptide receptor, glucagon receptor, glucagon-like peptide receptor, G-protein coupled estrogen receptor 1, KiSS1-derived peptide receptor, lysophosphatidic acid receptor, melanocortin 1 receptor, neuromedin B receptor, orexin receptor, prostaglandin E2 receptor, prostate-specific GPCR, Smoothened, sphingosine-1-phosphate receptor or thrombin receptor.
15. The tetravalent homodimeric antigen-binding protein of any claim 1 to 11, which is capable of specific binding to a tumour-associated antigen, such as but not limited to alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), MUC-1, CA-125, epithelial tumour antigen (ETA), epithelial cell adhesion molecule (EpCAM) or melanoma-associated antigen (MAGE).
16. The tetravalent homodimeric antigen-binding protein of any claim 1 to 11, which is capable of specific binding to a B-cell marker, such as but not limited to CD19, CD20, CD22 or CD38.
17. The tetravalent homodimeric antigen-binding protein of any claim 1 to 11, which is capable of specific binding to a receptor tyrosine kinase, such as but not limited to EGFR (HER1), HER2, HER3, c-Met, c-Kit or AXL.
18. The tetravalent homodimeric antigen-binding protein of any claim 1 to 11, which is capable of specific binding to a tumour-promoting growth factor, such as but not limited to vascular endothelial growth factor (VEGF) or angiopoietin-2.
19. The tetravalent homodimeric antigen-binding protein of any claim 1 to 11, which is capable of specific binding to a chemokine, such as but not limited to CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1 or CXCL8 (IL-8).
20. The tetravalent homodimeric antigen-binding protein of any claim 12 to 19, which is capable of specific binding to two different epitopes on the same target.
21. The tetravalent homodimeric antigen-binding protein of any claim 1 to 8, 10 and 12 to 19, which is a bispecific antibody capable of specific binding to (a) CD3 complex on T lymphocytes; or (b) CD28 co-stimulatory molecule on T lymphocytes; or (c) activating receptor FcγRIIIa (CD16a) on natural killer cells; or (d) NKG2D receptor on natural killer cells; or (e) inhibitory receptor FcγRIIb (CD32b) on B lymphocytes and myeloid dendritic cells.
22. A DNA sequence encoding the tetravalent homodimeric antigen-binding protein of any claim 1 to 21.
23. An expression vector comprising the DNA sequence of claim 22.
24. A host cell containing the expression vector of claim 23.
25. A pharmaceutical composition containing the tetravalent homodimeric antigen-binding protein of any claim 1 to 21, the DNA sequence of claim 22 or the expression vector of claim 23.
26. A pharmaceutical composition of claim 25 for use in diagnosis or patient stratification.
27. A diagnostic kit containing the tetravalent homodimeric antigen-binding protein of any claim 1 to 21 or the pharmaceutical composition of claim 25.
28. The tetravalent homodimeric antigen-binding protein of any claim 1 to 21 or the pharmaceutical composition of claim 25 for use in the treatment of (a) cancer; and/or (b) infectious diseases of viral, bacterial, fungal or prion origin; and/or (c) immune disorders; and/or (d) inflammatory diseases.

Images