WO2015063187A1 - Multivalent antigen-binding proteins - Google Patents

Abstract

The present disclosure relates to multivalent IgG-like antigen-binding proteins, polynucleotides encoding said antigen-binding proteins, expression vectors comprising these polynucleotides and prokaryotic or eukaryotic host cells comprising these polynucleotides or vectors. Furthermore, the disclosure relates to therapeutic and diagnostic uses of said molecules in the fields of oncology, inflammatory and autoimmune diseases.

Description

MULTIVALENT ANTIGEN-BINDING PROTEINS Technical Field

The present invention is directed to multivalent and multispecific 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 heteromeric; they comprise at least two different polypeptide chains that associate with each other 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 multivalent antigen-binding proteins of the present invention further comprise the constant domains of the antibody heavy (C_H2 and C_H3) and light chains (C-kappa or C-lambda) which allow stabilization of the multivalent antibody constructs and provide the antibody effector functions.

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).

The ability to generate therapeutic monoclonal antibodies (MAb) that are fully human (containing only proteins encoded by the human gene sequences) or humanized (comprising not more than 10% non-human amino acid sequences) or chimeric (comprising about 30% of non-human sequences) has been an important advance in immunotherapy. There are primarily three ways of generating therapeutic antibodies. The first approach is based on active immunization of animals (mice, rats, rabbits, camelids, etc.) followed by “chimerization”, i.e. combining the antigen-binding variable domains of the animal antibodies with the constant domains of human origin, or “humanization”, a kind of antibody engineering where the complementarity determining regions (CDR) of the selected antibodies of the animal origin are grafted into the human antibody frameworks. Currently, six chimeric and 19 humanized antibody therapeutics are approved in US and/or Europe for treatment of cancer and immune disorders; among them are the blockbusters rituximab (Rituxan™ / MabThera™), trastuzumab (Herceptin™) and bevacizumab (Avastin™) (Reichert, 2012, "Marketed therapeutic antibodies compendium", MAbs 4:413-5).

The second approach represents generation of fully human therapeutic antibodies by immunization of the transgenic (or trans-chromosomal) animals (mice, rats or rabbits) comprising human antibody encoding gene loci. This technique has been successfully used by a number of companies, such as Medarex (acquired by Bristol-Myers Squibb), Abgenix (acquired by Amgen), GenMab and Regeneron, and led to generation of eight therapeutic antibodies approved in US and/or Europe (Reichert, 2012, "Marketed therapeutic antibodies compendium", MAbs 4:413-5).

The third approach, originally introduced by the Cambridge Antibody Technology, CAT (now part of MedImmune / AstraZeneca) and followed by a number of companies, such as Dyax, BioInvent, Domantis (now part of GlaxoSmithKline), MorphoSys, etc., is to generate human antibodies in vitro by a technology known as “phage display”. In this latter approach, the entire spectrum of human antibody genes (either naïve or immune repertoire) can be cloned into a bacterial virus (a filamentous bacteriophage) in such a way that all possible human antibody proteins are individually “displayed” on the surface of bacteriophage particles, where each may be tested for binding to a target molecule. Such antibody gene collections are known as “phage display antibody libraries”. These antibody libraries are screened for binding to the disease-associated antigens, thus leading to generation of fully human therapeutic antibodies. Up-to-date, four therapeutic antibodies, including an anti-TNFα blockbuster adalimumab (Humira^®), have been approved in US and/or Europe.

Being highly specific, naturally evolved molecules, the antibodies are able to bind their soluble or cell-bound target antigens with high affinity and cause the pathogen inactivation or destruction of the tumour cells by antibody-dependent cellular cytotoxicity (ADCC), by antibody-dependent cellular (macrophage) phagocytosis (ADCP), by complement-dependent cytolysis (CDC), and/or by cross-linking the receptor followed by its internalization and apoptosis induction or by deprivation of the tumorigenic stimuli provided by the certain growth factors. Monoclonal antibodies are proven to be highly effective as drugs. They are selective, possess good CMC (Chemistry, Manufacturing and Control) properties and are produced at high yields in mammalian cells. In addition, the MAbs are stable and have long half-life in circulation. Both in liquid and solid tumours, antibodies have become an integral component of treatment regimens that have improved and extended the lives of cancer patients. For example, in hematologic cancers rituximab (Rituxan^® / MabThera^®) has become a component of the standard care in many non-Hodgkin’s lymphoma (NHL) subtypes due to the improved efficacy that it adds to chemotherapy regimens. In solid tumours, an anti-angiogenic antibody drug bevacizumab (Avastin^®) is becoming a standard of care in metastatic colorectal cancer (mCRC), non-squamous non-small cell lung cancer (NSCLC), metastatic breast cancer (mBC), metastatic renal cell carcinoma (mRCC), and glioblastoma as a first- or second-line therapy.

Despite these advances, however, there remains significant unmet need in cancer treatment. The MAbs are not generally effective as single agents against solid tumours and need to be administered in combination with chemo- and/or radiotherapy. Quite often, therapeutic efficacy is observed only in subsets of patients. For example, only about 25% of women with breast cancer respond to treatment with the blockbuster breast cancer drug Herceptin^®. Similarly, only 48% of NHL patients respond to Rituxan^®, which targets CD20. The clinical trials demonstrated that Avastin^® is ineffective for treatment of freshly operated colon cancer, and in advanced gastric cancer and advanced pancreatic cancer. There are also well documented severe side effects associated with Avastin^® treatment, such as gastrointestinal perforation (often fatal), high blood pressure, bleeding and wound healing complications, developing venous thromboembolism. No antibody therapies currently are available for the treatment of many other cancer types, including pancreatic, liver, bladder, or prostate cancers.

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), by 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).

The vast majority of the approved antibody drugs are made on the basis of naked immunoglobulins of IgG class. They are bivalent but monospecific, i.e. in most cases an antibody recognizes a single epitope on a particular antigen. Mutations in a tumour cell leading to changes in the epitope or even to disappearance of the epitope or the whole target molecule lead to generation of a tumour cell subpopulation that is resistant to treatment with this particular antibody.

To generate more potent antibodies that work better in combination or possibly as single agent therapy, 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, 2012, "Bispecific Antibodies and Immune Therapy Targeting", Drug Delivery in Oncology: From Basic Research to Cancer Therapy 2:441-82).

Bispecific antibodies (BsAb) are man-made proteins which are able binding two targets simultaneously. This property enables developing therapeutic strategies that are not possible with conventional monoclonal antibodies. For example, bispecific antibodies can override the natural specificity of an immunological effector cell for its target and redirect lysis towards a cell population it would otherwise ignore. Bispecific antibodies are designed either (1) to recruit the effector cells of the immune system (retargeting BsAb), (2) to block two or more targets simultaneously (BsAb of dual action), or (3) to provide higher selectivity of targeting cancer cells by simultaneous binding of two tumor-associated antigens (BsAb of enhanced selectivity) (Kiprijanov, 2012, "Bispecific Antibodies and Immune Therapy Targeting", Drug Delivery in Oncology: From Basic Research to Cancer Therapy 2:441-82).

Retargeting bispecific antibodies:

Retargeting BsAb can override the natural specificity of an immunological effector cell for its target and redirect lysis toward a cell population it would otherwise ignore. Immunological effector cells that can potentially be recruited by BsAbs include granulocytes, monocytes, macrophages, NK cells, and T cells. In contrast, human IgG1, which is the most widely used antibody isotype for tumour therapy, cannot recruit T-cells (the majority of which do not express Fc receptors), nor does it effectively trigger ADCC by polymorphonuclear neutrophils (PMNs), the most numerous cytotoxic effector cell population in humans. For cancer immunotherapy, the most desired effector cell populations are professional cell killers, such as CD56^dimCD16⁺ NK cells and CD8⁺ cytotoxic T-lymphocytes (CTLs). Both CTLs and NK cells contain preformed lytic granules comprising proteases of the granzyme family (especially granzyme A and B), perforin, and granulysin, and can kill several target cells in succession without killing themselves via the formation of the secretory synapses. Although the mechanism of apoptosis induction by granulocytes remains elusive, PMNs are also increasingly recognized as an important effector cell population for rejection of malignant tumours. Recruited PMNs produce several cytotoxic mediators, including reactive oxygen species, proteases, membrane-perforating agents, and soluble mediators of cell killing, such as tumour necrosis factor (TNF)-α, interleukin (IL)-1β, interferons, and antimicrobial peptides defensins, which are highly toxic against tumours. Myeloid cells infiltrate tumours engineered to secrete interleukins or chemokines in their microenvironment and play a key role in all of these cytokine-induced tumour rejections, often in cooperation with CD8⁺ T-lymphocytes.

To mediate redirected lysis, a BsAb must bind a target cell directly to a triggering molecule on the effector cell. The best-studied cytotoxic triggering receptors are multi-chain signalling complexes such as: (1) T-cell receptor (TCR) / CD3 complex on T-cells; (2) CD2 on T-cells and NK cells; (3) Fc receptors, such as low-affinity FcγRIIIa (CD16a) on NK cells, and high-affinity FcγRI (CD64) and FcαRI (CD89) expressed by monocytes, macrophages, and granulocytes; and (4) activating NK cell receptors, such as NKp46, NKp44, NKp30, NKp80 (KLR-F1), and NKG2D, which is also expressed on CD8⁺ T-cells. Due to the high affinity for IgG, all CD64 receptors appear to be occupied by serum IgGs. Therefore, a bispecific antibody targeting CD64 should bind to the outside of the Fc-binding domain of CD64.

It has been demonstrated that BsAbs can operate at lower concentrations than conventional antibodies and require lower target antigen expression. For example, a comparison of the recombinant CD19 × CD3 BsAb comprising two single-chain Fvs (scFvs) of antibody molecules connected in tandem by a peptide linker (tandem scFv or tascFv) with anti-CD20 chimeric MAb, rituximab, demonstrated 10⁵-fold difference in their cytotoxic efficacy (ED₅₀) in vitro (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).

Bispecific antibodies of dual action:

For most diseases, several mediators contribute to overall pathogenesis by either unique or overlapping mechanisms. The simultaneous blockade of several targets or targeting different pathogenic cell pools might therefore yield better therapeutic efficacy than inhibition of a single target. Designing of dual-action antibodies could help solve a major problem associated with monotherapy: cancer cells can become resistant to a single agent, mutating in ways that allow them to dodge the action of the drug. Having a single drug that can hit the cancer from multiple directions would simplify treatment and make it more efficient. A single antibody that could do the work of two is also attractive from a business perspective. It might cost half as much to manufacture as two separate antibodies, and the path to regulatory approval might also be shorter and less expensive, involving one set of clinical trials instead of multiple trials for two separate drugs in various dosage combinations.

Bispecific antibodies of enhanced selectivity:

The vast majority of tumour antigens are not really “tumour specific” (expressed exclusively on cancer cells); they are rather “tumour associated”. Although they are quite often overexpressed on tumour cells, these molecules are also present on normal cells and healthy tissues. For example, CD20, a target for the anti-lymphoma blockbuster rituximab (Rituxan^® / MabThera^®), is expressed on all B cells; the human EGFR (ErbB1, HER1), a target for cetuximab (Erbitux^®) and panitumumab (Vectibix^®) approved for treatment of colorectal cancer, is expressed on all epithelial tissues; HER2, a target for another bestseller drug, antibody trastuzumab (Herceptin^®), which is approved for treatment of HER2-positive metastatic breast cancer, is also present on heart and muscle cells. Lack of tumour specificity is a main reason for the adverse side effects associated with antibody therapy, such as acne-like skin rash in the case of Erbitux^® and Vectibix^®, and cardiotoxicity observed in some patients treated with Herceptin^®. However, there are combinations of tumour-associated antigens that can be found only on tumour cells and never on healthy tissues. For example, co-expression of CD38 and CD138 is thought to be exquisitely specific for myeloma cells (Stevenson, 2006, "CD38 as a therapeutic target", Mol Med 12:345-6), while CD38 alone is present on the surface of many immune cells (white blood cells), including CD4⁺ and CD8⁺ T-cells, and NK cells. Accordingly, CD138 is widely expressed on plasma cells. Combining two low/moderate-affinity antibodies (or antibody fragments) against each antigen can generate a dual-targeting bispecific molecule with high avidity for myeloma cells expressing both antigens, while binding weakly to cells expressing only one antigen. A similar approach can be proposed for targeting tumour cells co-expressing two members of the epidermal growth factor family of receptor tyrosine kinases, HER2 (ErbB2) and HER3 (ErbB3) (Robinson et al., 2008, "Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro", Br J Cancer 99:1415-25). Another example includes co-targeting CD5 (T-cell marker) and one of the B-cell markers, such as CD19, CD20, or CD23, that are co-expressed in most chronic lymphocytic leukaemia cells (Ahmadi et al., 2009, "Chronic lymphocytic leukemia: new concepts and emerging therapies", Curr Treat Options Oncol 10:16-32).

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. In addition, the IgG antibody light and heavy chains comprise the constant domains, C_L (C-kappa or C-lambda) and C_H1, C_H2 and C_H3, respectively. 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 tascFv (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 tascFv 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 the peptide linkers of more than 12 amino acids. 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 (Loffler 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; Loffler 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 EP1071752, US7112324 and WO9954440.

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 tascFv, 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 xenotransplantated 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 tascFv and the bispecific diabody formats mentioned above. 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 tascFv 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).

Although small recombinant BsAbs, such as diabodies and tascFv, may have an advantage in terms of tumour penetration, their size below kidney clearance threshold (around 60 kDa) leads to rapid elimination from the bloodstream by extravasation and glomerular filtration. This limitation could be overcome by generation of IgG-like bispecific molecules, which are too large to be easily filtered by the kidneys and comprise an Fc region binding to the neonatal Fc receptor (FcRn) that is responsible for antibody recycling and long serum half-life (Roopenian and Akilesh, 2007, "FcRn: the neonatal Fc receptor comes of age", Nat Rev Immunol 7:715-25). In addition, IgG-like BsAb are capable of supporting secondary immune functions, such as ADCC and CDC. However, production of bispecific IgG by co-expressing two different antibodies is inefficient due to mispairing of the antibody heavy and light chains (Marvin and Zhu, 2005, "Recombinant approaches to IgG-like bispecific antibodies", Acta Pharmacol Sin 26:649-58).

Thus, the technical problem underlying the present invention is to provide new multivalent IgG-like antigen-binding molecules that overcome the disadvantages of the bispecific antibodies of the prior art and to provide a general way to form a stable polypeptide molecules with at least four antigen-binding domains, which is monospecific or bispecific.

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 multivalent IgG-like 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 viruses, bacteria or fungi. Preferably, the multivalent antigen-binding proteins of the present invention can bind to at least two the same or different epitopes on the same or different antigen, wherein the said antigens are expressed on the same or different cells.

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. The present invention provides a general way to form a stable covalently linked antibody-like multivalent antigen-binding protein with at least four antigen-binding sites, which is monospecific or bispecific. Similar to the conventional IgG molecule, the multivalent antigen-binding protein of the present invention is formed by covalently linked two heavy and two light chains. However, unlike the conventional antibodies, each light chain comprises two variable domains, V_H and V_L, of the same or different specificity and one light chain constant domain, C_L (C-kappa or C-lambda). Accordingly, each heavy chain comprises two antibody variable domains, V_H and V_L, of the same or different specificity and three constant domains: antibody light chain constant domain, C_L (C-kappa or C-lambda), and the antibody heavy chain constant domains 2 (C_H2) and 3 (C_H3), wherein the C_L domain (C-kappa or C-lambda) and CH₂ domain are separated by the antibody hinge region. The presence of the antibody C_L domain (C-kappa or C-lambda) instead of the conventional heavy chain constant domain 1 (C_H1) within the said heavy chain provides better folding of the heavy chains and, thus, more efficient production of the multivalent antigen-binding protein of the present invention in different expression systems. Similar to the C_H1 domain, the C_L domain within the heavy chain is able to interact with the C_L domain within the light chain, thus providing formation of the stable heterotetrameric multivalent antigen-binding protein of the present invention.

The multivalent mono- or bispecific antigen-binding proteins of the present invention are expected to be very stable and have a higher antigen-binding capacity in comparison with the conventional mono- or bispecific antibodies. In addition, having a molecular weight of approximately 180-200 kDa (which is above the renal threshold) and an ability to bind FcRn (and, therefore, recycling) they should have favourable pharmacokinetics making them particularly useful for therapeutic purposes.

The present invention relates to a multivalent IgG-like antigen-binding protein formed by two light chains and two heavy chains, wherein

1. the light chain comprises two antibody variable domains, V_H and V_L, of the same or different specificity, followed by one antibody light chain constant domain, C_L (C-kappa or C-lambda);
2. the heavy chain comprises two antibody variable domains, V_H and V_L, of the same or different specificity, followed by one antibody light chain constant domain, C_L (C-kappa or C-lambda), by an antibody hinge region and by the antibody Fc region (C_H2 and C_H3 constant domains);
3. the antibody variable domains, V_H and V_L, of the light chain interact intermolecularly with the complementary V_L and V_H domains, respectively, of the heavy chain either in parallel (head-to-head) or in anti-parallel (head-to-tail) orientation to form the antigen-binding Fv modules (V_H/V_L pairs) pointing in opposite directions;
4. two heavy chains are covalently linked together via disulphide bonds formed by the cysteine residues located in the hinge region;
5. each light chain is covalently linked to the heavy chain via a disulphide bond formed by the cysteine residues located at the C-termini of the C-kappa (or C-lambda) domain within the heavy and light chain.

In a particularly preferred embodiment, the present invention relates to a multivalent antigen-binding protein characterized by the following feature:

1. the adjacent V_H and V_L domains of the light or heavy chains are derived from either the same or different antibody and are separated by the peptide linkers of less than 12 amino acids to prevent intramolecular pairing and to facilitate dimerization with the corresponding heavy or light chain.

A further preferred feature is that the antigen-binding V_H and V_L pairs are in V_H-to-V_L or in V_L-to-V_H orientation and are located in the N-terminal parts of the mature (devoid of the signal peptide) heavy and light chains.

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 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 polypeptide chains.

The term “pointing in opposite directions” means that the antigen-binding sites formed within the multivalent protein of the present invention have such orientation that they can simultaneously bind two antigens.

The multivalent antigen-binding polypeptides of the present invention can be prepared according to the standard methods and protocols. Preferably, the genes of the heavy or light polypeptide chain are prepared by ligation of the DNA sequences encoding the genes of the antibody variable (V_H and V_L) or constant (C-kappa or C-lambda, C_H2 and C_H3) 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 genes encoding the light and heavy chains of the IgG-like multivalent antigen-binding protein are ligated into a suitable expression vector for generation of the recombinant protein in the corresponding host cells, preferably mammalian cells.

The multivalent antigen-binding proteins of the present invention can comprise at least one further protein domain being 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.

Furthermore, the multivalent antigen-binding proteins of the present invention can be used to treat cancer as the antibody drug conjugates (ADC) or radioimmunoconjugates generated by chemical linking of the toxic payloads or radioactive compound (either directly or via a chelating agent). The multivalent antigen-binding proteins of the present invention can be conjugated with toxic chemotherapeutic drugs, such as e.g. maytansinoid drug DM1 or DM4, monomethyl auristatin E (MMAE) or auristatin F, calicheamicins and pyrrolobenzodiazepine dimers (Adair et al., 2012, "Antibody-drug conjugates - a perfect synergy", Expert Opin Biol Ther 12:1191-206). Different linkers that release the drug under acidic or reducing conditions or upon exposure to specific proteases are employed with this technology. The multivalent antigen-binding proteins of the present invention may be conjugated as described in the art.

In a preferred embodiment of the present invention, the multivalent antigen-binding proteins are monospecific. The order of the antibody-derived protein domains may give rise to the following light and heavy chains (see also Figures 1 and 2):

1 Anti-parallel (head-to-tail) orientation (Figure 1)

1-1 HC: V_L ^A-L1-V_H ^A-C_L-Hinge-C_H2-C_H3

LC: V_L ^A-L2-V_H ^A-C_L (L1, L2 < 12 aa)

1-2 HC: V_H ^A-L1-V_L ^A-C_L-Hinge-C_H2-C_H3

LC: V_H ^A-L2-V_L ^A-C_L (L1, L2 < 12 aa)

2 Parallel (head-to-head) orientation (Figure 2)

2-1 HC: V_L ^A-L1-V_H ^A-C_L-Hinge-C_H2-C_H3

LC: V_H ^A-L2-V_L ^A-C_L (L1, L2 < 12 aa)

2-2 HC: V_H ^A-L1-V_L ^A-C_L-Hinge-C_H2-C_H3

LC: V_L ^A-L2-V_H ^A-C_L (L1, L2 < 12 aa)

wherein “HC” is a heavy chain; “LC” is a light chain; “C_L” is an antibody light chain constant domain (C-kappa or C-lambda); L1 and L2 are the peptide linkers connecting the individual antibody variable domains (V_H and V_L) into a single-chain polypeptide; “A” is an antibody specificity.

In a further preferred embodiment of the present invention, the multivalent IgG-like antigen-binding proteins are bispecific. The order of the antibody-derived protein domains may give rise to the following light and heavy chains (see also Figures 3 and 4):

3 Anti-parallel (head-to-tail) orientation (Figure 3)

3-1 HC: V_L ^A-L1-V_H ^B-C_L-Hinge-C_H2-C_H3

LC: V_L ^B-L2-V_H ^A-C_L (L1, L2 < 12 aa)

3-2 HC: V_H ^A-L1-V_L ^B-C_L-Hinge-C_H2-C_H3

LC: V_H ^B-L2-V_L ^A-C_L (L1, L2 < 12 aa)

4 Parallel (head-to-head) orientation (Figure 4)

4-1 HC: V_L ^A-L1-V_H ^B-C_L-Hinge-C_H2-C_H3

LC: V_H ^A-L2-V_L ^B-C_L (L1, L2 < 12 aa)

4-2 HC: V_H ^A-L1-V_L ^B-C_L-Hinge-C_H2-C_H3

LC: V_L ^A-L2-V_H ^B-C_L (L1, L2 < 12 aa)

wherein “HC” is a heavy chain; “LC” is a light chain; “C_L” is an antibody light chain constant domain (C-kappa or C-lambda); L1 and L2 are the peptide linkers connecting the individual antibody variable domains (V_H and V_L) into a single-chain polypeptide; “A” and “B” are different antibody specificities.

In a further preferred embodiment of the present invention, the multivalent IgG-like antigen-binding proteins are multispecific. This is achieved by utilizing several different heavy and light chains comprising mutated C_L and hinge and/or C_H2 and/or C_H3 domains in the heavy chains and the mutated C_L domains in the light chains so that they are able to form stable interfaces only with the cognate mutated domains from the other heavy and light chains, respectively.

For the particular therapeutic applications, either heavy or light chain of the multivalent IgG-like antigen-binding protein 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.

Furthermore, the multivalent antigen-binding protein of the present invention can be Fc-engineered, i.e. may contain modified or mutated version of the Fc portion to provide, depending on the particular therapeutic application, stronger or weaker interaction with the corresponding Fc receptors or complement system and, therefore, modified effector functions, such as ADCC, ADCP, CDC and/or half-life in circulation (Hogarth and Pietersz, 2012, "Fc receptor-targeted therapies for the treatment of inflammation, cancer and beyond", Nat Rev Drug Discov 11:311-31).

In a preferred embodiment, the multivalent antibody-like antigen-binding protein 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 multivalent antibody-like antigen-binding protein 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 a further preferred embodiment, the multivalent antibody-like antigen-binding protein of the present invention is a bispecific antibody capable of specific binding to a tumour-associated antigen on the cancer cell and to the immune checkpoint antigens present of the immune effector cells, such as CTLA-4 (CD152), CD28, PD-1, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L, TIM3, or, alternatively, to the respective ligands present on the tumour cells, such as PD-L1 or PD-L2, CD80 or CD86, B7RP1, CD137L, OX40L, CD70, CD40, GAL9.

In an even more preferred embodiment, the multivalent antigen-binding protein 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 × c-Met; Axl × CTLA-4 (CD152); Axl × CXCR4; Axl × EGFR (HER1); Axl × HER2; Axl × HER3; Axl × PD-1; Axl × PD-L1; Axl × VEGF;
• CD123 × CD33; CD123 × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B);
• 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 × CD95 (APO-1); CD20 × CXCR4;
• CEA × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B); CEA × EpCAM; CEA × TNFα; CEA × VEGF;
• CXCR4 × VEGF;
• EGFR (HER1) × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B); EGFR (HER1) × CEA; EGFR (HER1) × c-Met; EGFR × CTLA-4 (CD152); EGFR (HER1) × EGFR (HER1) (biparatopic); EGFR (HER1) × EpCAM; EGFR (HER1) × HER2; EGFR (HER1) × HER3; EGFR (HER1) × IGF-1R; EGFR (HER1) × MerTK; EGFR (HER1) × PD-1; EGFR (HER1) × PD-L1; EGFR (HER1) × VEGF;
• EpCAM × CD3 (or CD16, or NKG2D, or NKp46, or NKp30, or CD32B); EpCAM × CCR4; EpCAM × CXCR4; EpCAM × VEGF;
• HER2 × Ang2; 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 × HER2 (biparatopic); HER2 × HER3; HER2 × VEGF;
• HER3 × CEA; HER3 × EpCAM; HER3 × VEGF.

In a further preferred embodiment, the multivalent antibody-like antigen-binding protein 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 multivalent antigen-binding protein, wherein the genes coding for the heavy and light chains are prepared by ligation of the DNA sequences encoding the genes of the antibody variable (V_H and V_L) or constant (C-kappa or C-lambda; C_H2, C_H3) 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 genes encoding the heavy and light chains of the antibody-like multivalent antigen-binding protein are ligated into suitable expression vectors for generation of the recombinant heteromeric protein (comprising two heavy and two light chains) in the corresponding host cells.

The present invention also relates to the DNA sequences encoding the multivalent antigen-binding proteins 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 multivalent antibody molecules. 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 , preferably, the 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. For certain therapeutic applications, the host cells with engineered glycosylation pathways may be utilized.

The present invention also relates to a pharmaceutical composition containing a multivalent 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, etc.); (c) treatment of infectious diseases caused by viruses, bacteria, fungi or which are prion-related.

A further object of the present invention is the use of a multivalent antigen-binding protein for the diagnostic purposes. The corresponding diagnostic tests are provided by the present invention, such as the kits comprising a multivalent antibody or a combination of several multivalent 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.

Brief Description of Drawings

F I GURE 1: Schematic representation of the domain organization in the heavy and light chains and a putative structure of a folded tetravalent monospecific antigen-binding protein of the present invention in an anti-parallel (head-to-tail) orientation of the Fv modules. “A” is an antibody specificity. N- and C-termini of the polypeptide chains are indicated as “N” and “C”, respectively. “H” indicates antibody hinge region.

FIGURE 2: Schematic representation of the domain organization in the heavy and light chains and a putative structure of a folded tetravalent monospecific antigen-binding protein of the present invention in a parallel (head-to-head) orientation of the Fv modules. “A” is an antibody specificity. N- and C-termini of the polypeptide chains are indicated as “N” and “C”, respectively. “H” indicates antibody hinge region.

FIGURE 3: Schematic representation of the domain organization in the heavy and light chains and a putative structure of a folded tetravalent bispecific antigen-binding protein of the present invention in an anti-parallel (head-to-tail) orientation of the Fv modules. “A” and “B” are different antibody epitope specificities. N- and C-termini of the polypeptide chains are indicated as “N” and “C”, respectively. “H” indicates antibody hinge region.

FIGURE 4: Schematic representation of the domain organization in the heavy and light chains and a putative structure of a folded tetravalent monospecific antigen-binding protein of the present invention in a parallel (head-to-head) orientation of the Fv modules. “A” and “B” are different antibody epitope specificities. N- and C-termini of the polypeptide chains are indicated as “N” and “C”, respectively. “H” indicates antibody hinge region.

Claims (31)

1. A multivalent antigen-binding protein comprising two heavy and two light chains, wherein (a) each light chain comprises two antibody variable domains, an antibody heavy chain variable domain VH and an antibody light chain variable domain VL, of the same or different specificity and one antibody light chain constant domain CL; (b) each heavy chain comprises two antibody variable domains, an antibody light chain variable domain VL and an antibody heavy chain variable domain VH, of the same or different specificity followed by one antibody light chain constant domain CL, by an antibody hinge region, by an antibody heavy chain constant domain 2 (CH2) and by an antibody heavy chain constant domain 3 (CH3); (c) the antibody variable domains, VH and VL, of the light chain interact intermolecularly with the complementary VL and VH domains, respectively, of the heavy chain either in parallel (head-to-head) or in anti-parallel (head-to-tail) orientation to form the antigen-binding Fv modules (VH/VL pairs) pointing in opposite directions.
2. The multivalent antigen-binding protein of claim 1, wherein two heavy chains are covalently linked together via disulphide bonds formed by the cysteine residues located in the hinge region.
3. The multivalent antigen-binding protein of any claim 1 to 2, wherein each light chain is covalently linked to the heavy chain via a disulphide bond formed by the cysteine residues located at the C-termini of the CL domains within the heavy and light chains.
4. The multivalent antigen-binding protein of any claim 1 to 3, wherein the antibody light chain constant domain CL is C-kappa or C-lambda.
5. The multivalent antigen-binding protein of any claim 1 to 4, wherein the adjacent VH and VL domains are separated by the peptide linkers of less than 12 amino acids to facilitate interchain interaction with the complementary VL and VH domains, respectively.
6. The multivalent antigen-binding protein of any claim 1 to 5, which is monospecific wherein the adjacent VH and VL domains are derived from the same antibody.
7. The multivalent antigen-binding protein molecule of any claim 1 to 5, which is bispecific wherein the adjacent VH and VL domains are derived from the different antibodies.
8. The multivalent antigen-binding protein of any claim 1 to 5, which is multispecific.
9. The multivalent multispecific antigen-binding protein of claim 8, wherein (a) it consists of two non-identical heavy and two non-identical light chains; (b) each heavy chain comprises mutated CL and/or hinge and/or CH2 and/or CH3 domains that are able to form stable interfaces only with the cognate mutated domains from the other heavy chain and only one light chain; (c) each light chain comprises mutated CL domain that is able to form stable interface only with the cognate mutated CL domain of only one heavy chain.
10. The multivalent antigen-binding protein of any claim 1 to 9, wherein the antigen-binding VH and VL pairs are in VH-to-VL or in VL-to-VH orientation.
11. The multivalent antigen-binding protein of any claim 1 to 10, wherein at least one polypeptide chain 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 multivalent antigen-binding protein of any claim 1 to 11, which may comprise modified or mutated version of the Fc portion to provide stronger or weaker interaction with the corresponding Fc receptors or complement system and, therefore, modified effector functions, such as ADCC, ADCP, CDC and/or half-life in circulation.
13. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a G-protein coupled receptor.
14. The multivalent antigen-binding protein of claim 13, where the G-protein coupled receptor is a chemokine receptor, such as CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or HCMV encoded chemokine receptor.
15. The multivalent antigen-binding protein of claim 13, 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 oestrogen 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.
16. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a tumour-associated antigen, such as alpha-fetoprotein (AFP), carcinoembryonic antigen (CEA), MUC-1, CA-125, epithelial tumour antigen (ETA), epithelial cell adhesion molecule (EpCAM) or melanoma-associated antigen (MAGE).
17. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a B-cell marker, such as CD19, CD20, CD22 or CD38.
18. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a receptor tyrosine kinase, such as EGFR, HER2, HER3, c-Met, c-Kit or AXL.
19. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a tumour-promoting growth factor, such as vascular endothelial growth factor (VEGF) or angiopoietin-2.
20. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a checkpoint antigen present of the immune effector cells, such as CTLA-4 (CD152), PD-1, ICOS, BTLA, KIR, LAG3, CD137, OX40, CD27, CD40L or TIM3.
21. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a checkpoint ligand, such as PD-L1, PD-L2, CD80, CD86, B7RP1, CD137L, OX40L, CD70, CD40 or GAL9.
22. The multivalent antigen-binding protein of any claim 1 to 12, which is capable of specific binding to a chemokine, such as CXCL10/IP-10, CXCL11/I-TAC, CXCL12/SDF-1 or CXCL8 (IL-8).
23. The multivalent antigen-binding protein of any claim 13 to 22, which is capable of specific binding to two different epitopes on the same target.
24. The multivalent antigen-binding protein of any claim 1 to 5, 7 to 23, which is a bispecific or multispecific 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.
25. A DNA sequence encoding the multivalent antigen-binding protein of any claim 1 to 24.
26. An expression vector comprising the DNA sequence of claim 25.
27. A host cell containing the expression vector of claim 26.
28. A pharmaceutical composition containing the tetravalent antigen-binding protein of any claim 1 to 24, the DNA sequence of claim 25 or the expression vector of claim 26.
29. A pharmaceutical composition of claim 28 for use in diagnosis or patient stratification.
30. A diagnostic kit containing the multivalent antigen-binding protein of any claim 1 to 24 or the pharmaceutical composition of claim 28.
31. The multivalent antigen-binding protein of any claim 1 to 24 or the pharmaceutical composition of claim 28 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.

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