GB2519786A - Multivalent antigen-binding protein molecules - Google Patents

Abstract

A multivalent antigen-binding protein molecule consisting of two heavy and two light chains is claimed, wherein (a) each light chain comprises a heavy chain variable domain (VH) and a light chain variable domain (VL), of the same or different specificity and one light chain constant domain (CL); (b) each heavy chain comprises a light chain variable domain (VL) and a heavy chain variable domain (VH), of the same or different specificity followed by one light chain constant domain (CL), by a hinge region, by a heavy chain constant domain 2 (CH2) and by a heavy chain constant domain 3 (CH3); (c) the light chain VH and VL domains interact intermolecularly with the complementary heavy chain VL and VH domains either in parallel (head-to-head) or in anti-parallel (head-to-tail) orientation to form the antigen-binding Fv modules pointing in opposite directions. Therapeutic and diagnostic uses of said molecules in oncology, inflammatory and autoimmune diseases is claimed.

Description

I

MULTIVALENT ANTIGEN-BINDING PROTEIN MOLECULES

1. FIELD OF THE INVENTION

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 inflammanory disorders. The domain-rearranged antibody molecules of the invention are heteromeric; they comprise at least two differeno polypetide 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 differeno cells. The individual poiypeptide chains of the DREAMs may be covalently linked through the covalent bonds, such as, but not limined, disulfide bonding of cysteine residues located within each polyneptide chain. In particular embodiments, the tetravalent molecules of the present invention further comprise the consoant domains of the antibody heavy (CL2 and C113) and light chains (C-kappa or C-lambda) which allow stabilization of the multivalent antibody constructs and provide the antibody effector functions.

2. BACKGROUND OF THE INVENTION

The recent clinical and commercial success of therapeutic antibodies has generated great interest in antibody-based therapeunics for hematological malignancies, solid tumors, autoimmune and inflammatory diseases (Rothe et al., 2008, "Therapeutic advances in rheumatology with the use of recombinant proteins", Nat din Pract Rheumatoi 4:605-14; Argyriou and Kalofonos, 2009, "Recent advances relating to the clinical application of naked monoclonal antibodies in solid tumors", Mol Ned 15:183-91; Chan and Carter, 2310, "Therapeutic antibodies for autcimmunity and inflammation", Nat Rev Immunol 10:301-16).

The ability to generate therapeutic monoclonal antibodies (NAb) 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 therapeucic antibodies. Ihe first approach is based on active immunizanion 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 oi human origin, or "humanization", a kind of antibody engineering where the compiementarity determining regions (CDR) of the selected antibodies of the animal origin are grafted into the human antibody frameworks. Currently, six chimeric and 15 humanized antibody therapeutics are approved in US and/or Europe for treanment of cancer and immune disorders; among them are the biockbusners rituximab (Rituxanm / MablheraTM) , trastuzumab (HerceptinTM) 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-ohromosomal) animals (mice, rats or rabbits) comprising human ancibody encoding gene looi. This technique has been successfully used by a number of companies, such as Nedarex (acquired by Bristol-Myers Squibb), Abgenix (acquired by Amgen), Cenl'dab and Regeneron, and led to generation of six therapeunio antibodies approved in US and/or Europe (Reichert, 2012, "Marketed therapeutic antibodies compendium", L'lAbs 4:413-5).

The third approach, originally introduced by the Cambridge Antibody Technology, CAT (now part of Medlmmune / Astrazeneca) and followed by a number of companies, such as Affitech, Biolnvenn, Domantis (now part of GiaxoSmithKline) , I4orphoSys, etc., is to generate human antibodies in vitro by a technology known as "phage display". In this iatter approach, the entire spectrum of human antibody genes (either naïve or immune repertoire) can be cloned into a bacterial virus (a filamenrous 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 tc 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 cc generation of fully human therapeutic antibodies. Up-to-date, three therapeutic antibodies, including an anti-TNFa blockbuscer adalimumab (HumiraTH) , have been approved in US and/cr 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 inactivacion or destructicn of the tumor cells by antibody-dependent cellular cytctoxicity (ADCC), by antibody-dependent cellular (macrophage) phagocytosis (ADCP) , by complement-dependent cytolysis (CDC) and/or by crcss-linking the receptor followed by its internalization and apoptosis induction or by deprivation of the tumorigenic stimuli provided by the certain growth factors.

Monoclonai antibodies are proven to be highly effective as drugs.

They are seleotive, possess good CMC (Chemistry, Manufacturing and Control) properties and are produoed at high yields in mammalian cells. In addition, the MAbs are stable and have long halt-life in circulation. Both in liquid and solid tumors, 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 improve!d efficacy that it adds to chemotherapy regimens. In solid tumors, an anti-angiogenic antibody drug bevacizumab (AvastinTM) is becoming a standard of care in metastatic colorectal cancer (mCRC), non-squamous non-small ccli lung cancer (NSCLC), metastatic breast cancer (mBC), metastatic renal cell carcinoma (mRCC), and glioblastoma as a first-or second-line therapy.

Despite These advanoes, however, there remains significant unmet need in cancer treatment. The MAbs are not generally effective as single agents against solid tumors and need to be administered in combination with chemo-and/or radiotherapy. Quite often, therapeucic efficacy is observed only in subsets of patients. Fcr example, only about 25% of women with breast cancer respond to treatmeno with the blockbuster breast cancer drug Herceptin.

Similarly, only 48% of NNL patients respond to RituxanlM, which targets CD2O. 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 AvastinrM treatment, such as gastrointestinal perforation (often fatal), high blood pressure, bleeding and wound healing complicarions, developing venous thromboembolism. No antibody therapies ourrently are available for the treatment of many other canoer types, inoluding gastric, pancreas, liver, bladder, or prostate cancers.

Malfunction ci naked immunoglobulins in some therapeutic seotings is accounted for by FcyRTITa (ODl6a) polymorphism (Cartron, 2009, "FCGR3A polymorphism story: a new piece of the puzzle", Leuk Lymphoma 50:1401-2), by interaction of antitumor antibodies with inhibitory Fo receptors (e.g., FoyRlIb) on myeloid cells (Clynes et al., 2000, "Inhibitory Pc receptors modulate in vivo cytotoxicity against tumor targets", Nat Med 6:443-6) and by differeno escape mechanisms developed by cancer cells to evade mortality (Baeuerle et al., 2003, "Bispecific antibodies for polyclonal f-cell engagement", Curr Opin Mol Ther 5:413-9).

The vast majority of the approved antibody drugs are made on the basis of naked immurioglobulins 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 tumor cell leading ro changes in the epitope or even to disappearance of the epitope or the whole target moleooie lead to generation of a tumor cell subpooulation 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 (Kipriyancv and Le Gail, 2004, "Recent advances in the generation cf bispecific antibodies for tumor immunotherapy", Curr Opin Drug Discov Devel 7:233-42; Kiprijanov, 2012, "Bispeoifio Antibodies and Lrcrnune 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 owo 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 ci two tumor-associated antigens (BsAb of enhanced selectivity) (Kirijanov, 2012, "Bispecific Antibodies and Izruuune 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 I cells. In contrast, human IgGi, which is the most widely used antibody isotype for tumor therapy, cannot recruit I-cells (the majority of which do not express Ec receptors), nor does it effectively trigger ADCC by nolymorphonuclear neutrophils (PMNs), the most numerous oytotoxic effector oell population in humans. For oancer immunotherapy, the most desired effeotor oell populations are professional cell killers, such as CD56'CDl6 NK cells and CD8 cytotoxic I-lymphocytes (CILs) . Both CTL5 and NK cells contain preformed lytic granules comprising proteases of the granzyme family (especially granzyme A and B) , perform, 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, P1'*lNs are also increasingly recognized as an important effector cell population for rejectIon of malignant tumors.

Recruited PF4Ns produce several cytotoxic mediators, including reactive oxygen species, proteases, membrane-perforating agents, and soluble mediators of cell killing, such as tumor necrosis factor (INF)-, interleukin (IL)-113, interferons, and antimicrobial peotides defensins, which are highly toxic against tumors. Myelcid cells infiltrate tumors engineered to secrere interleukins or chemckines in their microenvironment and play a key role in all of these cytokine-induced tumor rejections, often in cooperation with CD8 I-lymphocytes.

To mediane 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 signaling complexes such as: (1) T-cell receptor (ICR) / CD3 complex on I-cells; (2) CD2 on T-oells and NK cells; (3) Pc receptors, such as low-affinity FcyRIIIa (C016a) on NK cells, and high-affinity FcyRI (CD64) and FcaRI (0D89) expressed by monocytes, macrophages, and granulocytes; and (4) activating NK cell receptors, such as NKp46, NKp44, NKp3O, NKp8O (KLR-Fl), and NKG2E, which is also expressed on CD8' T-oells. Due to the high affinity fcr IgG, all CD64 receptors appear to be occupied by serum IgGs. Therefore, a bispeoific antibody targeting CD64 should bind to the outside of the Fo-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 x CD3 EsAb comprising two single-chain Fvs (scFvs) of antibody molecules oonnected in tandem by a peptide linker (randem scFv or tasofv) with anti-CD2O chimenic MAb, nituximab, demonstrated 105-fold difference in their cytotoxic efficacy (ED) 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", Tnt J Canoer 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 blookade of several targets or targeting different pathogenic cell pools might therefore yield better therapeucio efficacy than inhibition of a singie 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 efficienc. A single antibody that could do the work of two is also attractive from a business perspective. It might cost half as much no manufacture as two separate antibodies, and the path to regulatory apuroval 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 majcrity of tumor antigens are not really "tumor specific" (expressed exclusively on cancer cells) ; they are rather "rumor associated". Although they are quite often overexpressed on tumor cells, these molecules are also present cm normal cells and healthy tissues. For example, CD2O, a target for the anti-lymphoma blockbuster rituximab (Rituxanm / MablheraTM), is expressed on all B cells; the human EGER (ErbBl, HER1), a target for cetuximab (ErbituxrM) and panitumumab (VectibixT) 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 treatmenc of HER2-positive metastatic breast cancer, is also present on heart and muscle cells. Lack of tumor specificity is a main reason for the adverse side effects associated with ancibody therapy, such as acne-like skin rash in the case of Erbitux and Vectibix, and cardiotcxicity observed in some patients treated with HerceptinlN. However, there are combinations of tumor-associated antigens that can be found only on tumor 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 12:3'5- 6), while CD3S alone is present on the surface of many immune cells (white blood cells), including CD4 and CDB' I-cells, and NK cells. Accordingly, CD138 is widely expressed on plasma cells.

Combining two low/moderate-affinity antibodies (or antibody fragrr.ents) against each antigen can generate a dual-targeting bispecific molecule with high avidity for myeloma cells expressing bcth antigens, while binding weakly to cells expressing only one antigen. A similar approach can be proposed for targeting tumor cells cc-expressing two members of the epidermal grcwth 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 cc-targeting CD5 CT-cell marker) and one of the B-cell markers, such as CD19, CD2O, or CE23, that are co-expressed in most chronic lymphocytic leukemia 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 tricma-made anti-human EpCAV / anti-human 0D3 half-mouse/half-rat bispecific antibody catumaxomab (RemovabTM) 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 Bid Ther 10:1259- 69) . However, a major limitation of the bispecific antibodies produced by hybrid hybridomas (guadromas) (Milatein and Cuello, 1983, "Hybrid hybridcmas and their use in immunohistcchemistry", Nature 305:537-40) or by using a trioma (cross-species hybridoma) technology (Mooikat et al., 1997, "Trioma-based vaccination against B-cell lyruphoma confers long-lasting tumor immunity", Cancer Res 57:2346-9) is their immunogenicity. Repeated doses of rodent antibodies elicit an anti-imnunoglobulin antibody response, which compromises therapy with bispecific antibody. For example, roughly one third of the patients treated with the trioma-made antibodies develop immune reaotion to mouse or rat protein (HAMA/HARA response) (Kiewe and Thiel, 2008, "Ertumaxomab: a trifunctional antibody for breast oancer treatment", Expert Opin Investig Drugs 17:1553-8) An intaco unmodified antibody of TgG class is a heterotetramer comprising two heavy and two light poiypeptide chains. The N-terminal parts of the heavy and light chain, the so-called variable (V) domains (Vt: and \:, respectively) , form the antigen-binding ffragment variable' (Fv) of an antibody. In addition, the IgG antibody light and heavy chains comprise the constant domains, C (C-kappa or C-lambda) and C41, C2 and CH3, respectively. The domain architecture of antibodies and the advances in recombinant DNA technology provide an opportuniny to develop methods for engineering and producing bispecific antibodies exclusively from the antigen-binding (Fv) antibody fragirnts (Kipriyanov and Ic Gail, 2004, "Recent advances in the generation of bispecific antibodies for tumor immunotherapy", Curr Opin Drug Discov Devel 7:233-42; Chaises 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 lin:cer was introduced between The variable domains of the antibody heavy and lighu chain wiTh the formation of the so-called single-chain (so) 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 coil", Proc Natl Acad Sci U S A 85:5879-83) Two soEv-based bispeoifio antibody formats have been intensively studied, tandem soFvs or tasoFv (Mack et al., 1995, "A small bispecific antibody construct expressed as a functional single-chain molecule with high tumor cell cytotoxicity", Proc Nati Acad Sci U S A 92:7021-5) and diabodies (Holliger et al., 1993, ""Diabodies": small bivalent and bispecific antibody fragments", Proc Nati Acad Sci U S A 90:6444-8; Johnson et al., 2010, "Effector cell recruitment with novel Ev-based dual-affinity re- targeting protein leads to potent tumor cytolysis and in vivo B-cell depletion", J Mci Biol 399:436-49) . In a tascFv approach, the individual protein domains, such as heavy and light chain antibody variable domains (V-1 and VL, respectively) from two antibodies of different specificity, are fused together as a single polypeptide chain in an order, e.g., VLA_VEA_VE_VI (where A and B indicate different specific±ties), and the functional antigen-binding Fr 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-celi engager (BITEm) antibodies which showed high potency in killing tumor cells by 7-cell recruitment both in vitro (Loftier et al., 2000, "A recombinant bispecific single- chain antibody, 0D19 x CD3, induces rapid and high lymphoma-directed cytotoxicity by unstimulated 7 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", Tnt J Cancer 100:690-7; Loffler et al., 2003, "Efficient elimination of chronic lymphocytic leukaemia B cells by autologous 7 cells with a bispecific anti-CD19/anti-CD3 single-chain antibody construct", Leukemia 17:900-9) and in animal models (Dreier et al., 2003, "7 cell costimulus-independent and very efficacious inhibition of tumor growth in mice bearing subcutaneous or leukemio human B cell lymphoma xenografts by a CDl9-/CD3-bispecific single-chain antibody construct", J Immuncl 170:4397-402), and demonstrated promising results in clinical trials (Bargcu 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 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, U37112324 and W09954440.

In the second method, the recombinant bispecific molecules are formed by non-covalent association of two hybrid scFvs, e.g., such as VII7'VL and Vi1-ViI, each comprising V11 and Vi. domains of differenu specificity (A and B, respectively) , separated by a short peptide linker (<12 amino acids) that prevents intramolecular Vp/V-pairing, thus giving a four domain bispecific diabody (Kipriyanov et al., 1998, "Bispecific CD3 x CDl9 diabody for T cell-mediated lysis of malignant human B cells", mt j Cancer 77:763-72). In general, diabodies are well folded molecules and, unlike tasclv, 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; Cochiovius et al., 2000, "Treatment of human B cell lymphoma xenografts with a CD3 x CDJ9 diabody and T cells", J Immunol 165:888-95) . They have also demonstrated high activity in recruitment of either I cells or NK cells to kill tumor cells both in vitro and in animal models (Kipniyanov et al., 1998, "Bispecific CD3 x C519 diabody for T cell-mediated lysis of maiignant human B cells", :nt 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-B; Coohlovius et al., 2000, !!Treatmert of human B cell lymphorna xenografta 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 0D3 and CDl9 x 0D16 diabodies in a preclinical model of non-Hodgkin's lymphoma", J Imirunol 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 and VL 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 cascFv 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 partic-niar 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 B1TEIM 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, suoh as diabodies and tascfv, may have an advantage in terms of tumor 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 TgC-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 halt-life (Rocpenian and Akiiesh, 2007, "FcRn: the neonatal Fc receptor comes of age", Nat Rev Immunol 7:715-25). In addition, IgG-like BsAb are capable of supporcing secondary immune functions, such as ADCC and CDC. However, production of bispeoifio igo by co-expressing two different antibodies is inefficient due to mispairing of the antibody heavy and lighu 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 TgG-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 moncspecific or bispeoific.

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

3. DESCRIPTION OF THE INVENTION

The present invention relates to the multivalent IgC-iike antigen-binding nolypeptides and to their use in the treatment of a variety of diseases and disorders inoluding canoer, autoimmune disorders, allergy, inflammatory disorders and infectious diseases caused by viruses, bacteria or fungi. Preferably, nbc multivalent antigen-binding protein molecules of the presenu 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 oomplementarity of the cognate Vh and VL domains derived from the same antibody and their abiiity to form heterodimers. Although in the most cases stability of a single Fv module (non-covalent V-/Vj heterodiiner) is low, with a dissociation constant (K-i in the range of 1-10 pSI (Giockshuber et al., 1990, "A comparison of strategies to stabilize immunoglobulin Fv-fragments", Biochemistry 29:1362-7), the single-chain polypeptides comprising several V and V 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 molecule with at least four antigen-binding sites, which is monospeoific or bispecific. Similar to the conventicnal IgG molecule, the multivalent protein molecule 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, VH and 17L, of the same or differenc specificity and one light chain constant domain, C (C-kappa or C-lambda) . Accordingly, each heavy chain comprises two antibody variable domains, V1 and Vt, of the same or different specificity and three constant domains: antibody light chain constant domain, CT! (C-kappa or C-lambda) and the antibody heavy chain constant domains 2 (C112) and 3 (C113), wherein the CL domain (C-kappa or C-lambda) and CU2 domain are separated by the antibody hinge region. The presence of the antibody Cr domain (C-kappa or C-lambda) instead of the conventional heavy chain constant domain 1 (CE1) within the said heavy chain provides better folding of the heavy chains and, thus, more efficient production of the multivalent antigen-binding protein molecule of the present invention in different expression systems. Similar to the C111 domain, the C1. domain within the heavy chain is able to interact with the Cr domain within the light chain, thus providing formation of the stable heteroteorameric multivalent molecule of the present invention.

The multivalent mono-or bispecific antigen-binding protein molecules of the present invention are expected to be very stable and have a higher antigen-binding oapaoity in comparison wiuh the conventional mono-or bispecifio antibodies, Tn 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 favorable pharmacokinetics making them paroicularly useful for therapeutic purposes.

The present invention relates to a multivalent IgG-iike antigen-binding protein molecule formed by two light chains and two heavy chains, wherein (a) the light chain comprises two antibody variable domains, V and Vt, of the same or different specificity, followed by one antibody light chain constant domain, C-(C-kappa or C-lambda); (b) the heavy chain comprises two antibody variable domains, V11 and Vt, of the same or different specificity, followed by one antibody light chain constant domain, C (C-kappa or C-lambda), by an antibody hinge region and by the antibody Fc region (C112 and C3 constant domains); (o) the antibody variable domains, VE and V, of the light chain interact intermolecularly with the complementary \J 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 (Vu/Vt pairs) pointing in opposite directions; (d) two heavy chains are covalently linked nogether via disulfide bonds formed by the cysteine residues located in the hinge region; (e) each light chain is covalently linked to the heavy chain via a disulfide 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 parcicularly preferred embodiment, the present invention relates o a multivalent antibody-like molecule characterized by the following feature: the adjacent 1⁄4 and VL 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 Vu and Vj, pairs are in V-T-tO-VT, or in Vt-tO-VT-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 oapable of conneoting two antibody domains with its length depending on the kinds of domains to be conneoted. 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 VH and VL domains belonging to the same polypeptide chain with the formation of functional antigen-binding site.

The term "intermolecularly" means Interaction of the cognate Vu and VL domains, which belong to different polypeptide chains.

The multivalent antigen-binding polypeptides of the present invention can be prepared according to the standard methods and protccols. Preferably, the genes cf the heavy or light polypeptide ohain are prepared by ligation of the DNA seguenoes enooding the genes of the antibody variable (V-1 and VL) or constant (0-kappa or 0-lambda, Cu2 and Cu3) domains. The genes of the antibody domains are generated either by chemical synthesis or are produced by a polymerase chain reaction (FOR) 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 lymphocyces, spleens and/or tcnsils) . The assembled genes enooding the light and heavy chains of the IgG-like multivalent molecule 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 molecules 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 dcmain 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 nbc 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 nbc proteinases.

Furthermore, the multivalent antigen-binding molecules of the present invention can be used to treat cancer as the antibody drug conjugates (ADC) or radioimmuncconjugates generated by chemical linking of the toxic payloads or radioactive compound (either directly or via a chelating agent) . The multivalent antigen-binding molecules of the present invention can be conjugated with toxic chemotherapeutic drugs, such as e.g. maytansinoid drug DM1 or DM4, monomethyl auristatin E (t'24AE) or auristatin F, and calicheamicins (Adair et al., 2012, "Antibody-drug conjugates -a perfect synergy", Expert Opin Bioi 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 multivalenc antigen-binding molecules of the present invention may be conjugated as described in the art.

In a preferred embodiment of the present invention, the multivalent antigen-binding molecules 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 HO: VJ-Li-Vi-O-Hinge-C2-03 LC: Vj-L2-V-C. (Li, L2 < 12 an) i-2 HO: VH-Li-vL-0-Hinge-C2-03 LO: VflA_L2_VLA_C (Li, L2 < 12 aa) 2 Parallel (head-to-head) orientation (Figure 2) 2-1 HO: V-:-Li-V-H-C--Hinge-C2-C3 LO: VflA_L2_VL_O (Li, L2 < 12 aa) 2-2 HO: Vfl-Li-vL-O-Hinge-C2-O2 LO: \TA_L2_v1A_O (Li, L2 < 12 an) wherein "HO" is a heavy chain; "LO" is a light chain; CL" is an antibody light chain constant domain (C-kappa or C-lambda); Li and L2 are the peptide linkers connecting the individual ancibody variable domains (V[ and V-) into a single-chain polypeptide; "A" is an annibody specificity.

In a furTher preferred embodiment of the present invention, the multivalent TgG-like antigen-binding molecules 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 HO: LO: Vf-L2-V-i-CL (Li, L2 < 12 an) 3-2 HO: \THA_Li_VLb_0L_Hinge_C2_0:3 LO: VH_L2_VTA_C, (Li, L2 < 12 aa) 4 Parallel (head-to-head) orientation (Figure 4) 4-1 HO: VJ-Li-V-0-Hinge-C2-03 LO: VHA_L2_VLB_OL (Li, L2 < 12 aa) 4-2 HO: irA_Li_V LO: Vj-L2-Vi'-O (Li, L2 < 12 ac) wherein "HO" is a heavy chain; 1LO" is a light chain; OL" is an antibody light chain constant domain (C-kappa or C-lambda) ; Li and L2 are the peptide linkers connecting the individual ancibody variable domains (V1-and V-) 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 molecules are multispecific.

This is achieved by utilizing several different heavy and light chains comprising mutated CL and hinge and/or C2 and/or C3 domains in the heavy chains and the mutated OL 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-Iike molecule 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, oyclosporine, etc.), an anti-neoplastic agent (e.g., monomemyl 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 labeled.

Furthermore, the multivalent antigen-binding molecule 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 multivaient antibody-like molecule 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 tumor-associated antigen (such as Axi, CD19, CD2O, CEA, EGER, EpCAM, FGFR, HER2, HER3, etc.), or a tumor-promocing growth factor (e.g., VEGE, angiopoietin-2, etc.), or a chemokine (e.g., CXCL1O/IP-i0, CXCL11/I-TAC, CXCL12/SDF-l, etc.).

In a furTher preferred embodiment, the multivalent antibody-like molecule of the present invention is a monospecific biparatopic antibody capable of specific binding to the different epitopes cn the same antigen from the group of GPCR, preferably the chemokine receptor (e.g., CCR4, CCR5, CXCR3, CXCR4, etc.), or tumor-associated antigens (such as Axl, CD19, CD2O, CEA, EGFR, EpCAM, FGFR, HER2, HER3, etc.), or tumor-promoting growth factors (e.g., VEGF, angiopoietin-2, etc.), or the chemokines (e.g., CXCL1O/IP- 10, CXCL11/I-IAC, CXCL12/SDF-1, etc.).

In an even more preferred embodiment, the multivalent antibody-like molecule of the present invention is a bispeoific antibody capable of speoifio binding to the following antigen pairs present on the same or different cells: -Axi x CD3 (or CD16, or NKG2D, or NKp46, or NKp3O, or CD32B); -Axi x c-Met; -Axi x CXCR4; -Axi x EC-FR (HER1); -Axi x HER2; -Axl x HER3; -Axl x VEGF; -CD123 x CD33; -CD123 x CD3 (or CD16, or NKG2D, or NKp46, or NKp3O, or CD32B); -CD19 CD3 (or CD16, or NKG2I, or NKp46, or NKp3O, or CD32B); -CD2O x CD3 (or CD16, or NKG2D, or NKp46, or NKp3O, or CD32B); -0D19 x CD2O; -CD19 x CD22; -CD2O x CD22; -CD2O x CD95 (APO-1); -CD2O CXCR4; -CEA x CD3 (or CD16, or NKG2D, or NKp46, or NKp3O, or CD32B); -CEA x EpCAM; -CEA x TNFc; -CEA x VEGF; -CXCR4 x VEGF; -EGER (HER1) x CD3 (or CD16, or NKG2D, or NKp46, or NKp3O, or CD32B); -EGER (HER1) x CEA; -EGER (HER1) x c-lVJet; -EGER (HER1) x EGFR (HER1) (biparatopic) -EGER (HER1) x EpCAM; -EGER (HER1) x HER2; -EGER (HER1) x HER3; -EGF'R (HER1) x IGF-iR; -EGFR (HER1) x t4erTK; -EGFR (HER1) x VEGF; -EpCAt4 x CD3 (or CDIL6, or NKG2D, or NKp46, or NKp3O, or CD32B); -EpCA4 x CCR4; -EpCAI4 x CXCR4, -EpCAt4 x VEGF; -HER2 x Ang2; -HER2 CD3 (or CD16, or NKG29, or NKp46, or NKp3O, or CD32B); -HER2 x CEA; -HER2 x CXCR4 (or CCR4, or CCR7, or SiP-); -HER2 x EpOAM; -HER2 x HER2 (biparatopic); -HER2 x HER3; -HER2 x VEGE; -HER3 x CEA; -HER3 x EpCAM; -HER3 x VEGE.

In a furTher preferred ernbodiment, the multivalent antibody-like molecule of the oresent 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., VEGE, angiopoietin-2, human serum albumin, etc.) Another object of the present invention is a process for the preparation of a multivalent antigen-binding molecule, 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 (VH and Vj) or constant (C-kappa or C-lambda; C2, Cr3) 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, peripherai blood lymphocytes, spleens, tonsils) . The assembled genes encoding the heavy and light chains of the antibody-like multivalent molecule 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 protein molecules 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 icr pronagation and expression of the DNA seguences 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 (Saccharornyces, 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 D140, E366, etc., and, preferably, the mammalian cells, such as Chinese Hamster Ovary (CUD) , human embryonic kidney cells (HEK-293), PER.C6, etc., stably or transienciy 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 convencional 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 (IF), subcutaneous (SC), intramuscular (TM), intravitreal (IVT), intradermal (TD) 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 (hematological, solid, metastacic, minimal residual disease) ; (b) treatment of inflammatory and immune disorders (such as rheumatoid arthritis, systemic lupus erythemacosus, 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 molecule for the diagnostic purposes.

The corresponding diagnostic tests are provided by the present invention, such as the kits comprising a multivalent antibody or a coiftination of several multivalent antibodies of the present invention. The compound of the present invention can be detectably labeled 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) GyrolabtN immunoassay platform or medical imaging.

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

4. BRIEF DESCRIETTON OF THE DRAWINGS FIGURE 1: Schematic representation of the domain organization in the heavy and light chains and a putative structure of a folded tetravalent monospecific antigen-binding molecule of the present invention in an anti-parallel (head-to-tail) orientation of the Fv modules. "A" is an antibody specificity. N-and C-termlni 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 molecule of the present invention in a parallel (head-to-head) orientation of the Tv modules. "A" is an antibody specificity. N-and 0-termini of the polypeptide chains are indicated as "N" and "0", respectively.

"H" indicates antibody hinge region.

FIGURE 3: Schematic representation of the domain organization in the heavy and light ohains and a putative structure of a folded tetravalent bispecific antigen-binding molecule 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 molecule of the present invention in a parallel (head-to-head) orientation of the Ev 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 (29)

1. CLAIMS1. A multivalent antigen-binding protein molecule consisting of two heavy and two light chains, wherein (a) each light chain comprises two antibody varlabie 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) crientation to form the antigen-binding Fv modules (VH/VL pairs) pointing in opposite directions.
2. 2. The multivalent antigen-binding prctein molecule of claim 1, wherein two heavy chains are covalently linked together via disulfide bonds formed by the cysteine residues located in the hinge region.
3. 3. The multivalent antigen-binding prctein molecule of any claim I to 2, wherein each light chain is covalently linked to the heavy chain via a disulfide bond formed by the cysteine residues located at the C-termini of the CL domains within the heavy and light chains.
4. 4. The multivalent antigen-binding protein molecule of any claim I to 3, wherein the antibody light chain constant domain CL is C-kappa or C-lambda.
5. 5. The multivalent antigen-binding protein molecule of any claim I 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. 6. The multivalent antigen-binding protein molecule of any claim I to 5, which is moncspecifio (i.e., the adjacent VH and Vt domains are derived from the same antibody)
7. 7. The multivalent antigen-binding protein molecule of any claim I to 5, which is bispecific (i.e., the adjacent V and Vt domains are derived from the different antibodies)
8. 8. The multivalent antigen-binding protein molecule of any claim I to 5, which is multispeoific.
9. 9. The multivalent multispecific antigen-binding protein molecule of claim 8, wherein (a) it consists of two non-identical heavy and two non-idennical light chains; (b) each heavy chain comprises mutated CL and/or hince 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; Ko) each light chain comprises mutated CL domain that is able:o form stable interface only with the cognate mutated CL domain of only one heavy chain.
10. 10. The multivalent antigen-binding protein molecule of any claim 1 to 9, wherein the antigen-binding IT and IT pairs are in VH-to-VL or in \7L-to-VH orientation.
11. 11. The multivalent antigen-binding protein molecule of any claim 1 to 10, wherein at least one polypeptide chain is covalently or non-covalently linked to a biologically active protein, a chernotherapeutic agent, an anti-neoplastic agent, a peptide, a protease, or radioactively labeled.
12. 12. The multivalent antigen-binding protein molecule of any claim 1 to 11, which is Fc-engineered, i.e. may contain modified or mutated version of the Fc portion to provide stronger or weaker interaction with the corresponding Fc receptors or complement system and, therefcre, modified effector func:ions, such as ADCC, ADCP, CDC and/or half-life in circulation.
13. 13. The multivalent antigen-binding protein molecule of any claim 1 to 12, which is capable of specific binding to a G-protein coupled receptor.
14. 14. The multivalent antigen-binding protein moleoule of claim 13, where the G-protein coupled receptor is a chemokine receptor, such as CCR3, CCR4, 00R5, 00R6, CCR7, CCR8, CXCR1, CXCR2, CXCR3, CXCR4, CXCR6, CXCR7 or HCMV encoded chemokine receptor.
15. 15. The multivalent antigen-binding protein molecule of claim 13, where the G-protein coupled receptor is angiotensin II receptor AT1, beta-adrenergic receptor, bradyklnin receptor, cannabinoid receptor, cholecystokinin A receptor, endothelin 1 reoeptor, free fatty acid receptor, Frizzled, gastric-inhibitory-peptide-receptor, gastrin-releasing peptide receptor, glucagon receptor, glucagon-like peptide receptor, 0-protein coupled estrogen receptor 1, KiSSl-derived peptide receptor, lysophosphatidic acid receptor, melanocortin 1 receptor, neuromedin B receptor, orexin receptor, prostaglandin E2 receptor, prostate-specific 09CR, Smoothened, sphingosine- 1-phosphate receptor or thrombin receptor.
16. 16. The multivalent antigen-binding protein molecule of any claim 1 to 12, which is capable of specific binding to a tumor-associated antigen, such as alphafetoprotein (ATP) carcinoembryonic antigen (CEA), MUC-1, CA-125, epithelial tumor antigen (ETA), epithelial cell adhesion molecule (EpCAM) or melanoma-associated antigen (MACE)
17. 17. The multivalent antigen-binding protein molecule of any claim 1 to 12, which is capable of specifio binding to a B-cell marker, such as CD19, CD2O, CD22 or CD3B.
18. 18. The multivalent antigen-binding protein molecule of any claim 1 to 12, which is capable of specific binding to a receptor tyrosine kinase, such as EGER, HER2, HER3, c-Net, c-Kit or AXL.
19. 19. The multivalent antigen-binding protein molecule of any claim 1 to 12, which is capable of specific binding to a tumor-promo:ing growth factor, such as vascular endothelial growth factor (VEGF) or angiopcietin-2.
20. 20. The multivalent antigen-binding protein molecule of any claim 1 to 12, which is capable of specific binding to a chemokine, such as OXCL1O/IP-lO, CXCL11/I-TAC, CXCL12/SDF-1 or CXCL8 (IL-B)
21. 21. The multivalent antigen-binding protein molecule of any claim 13:0 20, which is capable of specific binding to two different epitopes on the same target.
22. 22. The multivalent antigen-binding protein molecule of any claim 1 to 5, 7 to 21, which is a bispecific or multispecific antibody capable of specific binding to: (a) CD3 complex on T lymphocytes; or (b) CD28 co-stirnulatory molecule on T lymphocytes; or (c) activating receptor EcyRlila (CD16a) on natural killer cells; or (d) NKG2D receptor on natural kiLler cells; or (e) inhibitory receptor FcyRIIb (CD32b) on B lymphocy:es and myeloid dendritic cells.
23. 23. A DNA sequence encoding the multivalent antigen-binding protein molecule of any claim 1 to 22.
24. 24. An expression vector comprising the DNA sequence of claim 23.
25. 25. A host rell containing the expression vector of claim 24.
26. 26. A pharmaceutical composition containing the tetravalent antigen-binding protein molecule of any claim 1 to 22, the DNA sequence of claim 23 or the expression vector of claim 24.
27. 27. A pharmaceutical composition of claim 26 for use in diagnosis or patient stratification.
28. 28. A diagnostic kit containing the multivalent antigen-binding protein molecule of any claim 1 to 22 or the pharmaceutical composition of claim 26.
29. 29. The multivalent antigen-binding protein molecule of any claim 1 to 22 or the pharmaceutical composition of claim 26 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.