BR112021005670A2 - tetrameric her2 binding polypeptides - Google Patents

Abstract

A tetrameric polypeptide comprising a first polypeptide chain comprising a first VL antigen binding domain and a first constant CL domain, a second polypeptide chain comprising a first VH antigen binding domain, a first constant CH1 domain, a first constant CH2 domain, and a first constant domain CH3, a first linker binding to a D4 epitope of HER2 linked to the N-terminus of said first VL antigen binding domain or said first VH antigen binding domain by a first interdomain amino acid linker, a third chain A polypeptide comprising a second VL antigen binding domain and a second CL constant domain, a fourth polypeptide chain comprising a second VH antigen binding domain, a second constant CH1 domain, a second constant CH2 domain, and a second constant CH3 domain, and a third ligand that binds to a terminal-bound HER2 D4 epitope N of said second VL antigen binding domain or said second VH antigen binding domain by a second interdomain amino acid linker, wherein the VL antigen binding domains and the VH antigen binding domains together constitute a second ligand and a fourth ligand that binds to a D1 epitope of HER2. The invention also relates to a tetrameric polypeptide for use in a method for the prevention or treatment of a malignant neoplastic disease, an isolated nucleic acid and a host cell for expressing the polypeptide, and a method for obtaining the polypeptide.

Description

Patent Descriptive Report for "HER-2 BINDING TETRAMERIC POLYPEPTIDES".

The present invention relates to a tetrameric polypeptide that has two binding sites to the D1 epitope of HER?2 and two binding sites to the D4 epitope of HER?2. Background of the invention

[0002] Members of the HER family of receptor tyrosine kinases are important mediators of cell growth, differentiation, migration and survival. The receptor family includes four distinct members, including the epidermal growth factor receptor (EGFR, ErbB1 or HER1), HER2 (ErbB2 or p185<neu>), HER3 (ErbB3) and HER4 (ErbB4). Members of the EGFR family are single-chain modular glycoproteins closely related to an extracellular ligand binding region, a single transmembrane domain, and an intracellular tyrosine kinase, followed by specific phosphorylation sites that are important for anchoring downstream signaling proteins .

[0003] The extracellular regions of the HER receptor family contain two homologous ligand binding domains (domains 1 and 3) and two cysteine-rich domains (domains 2 and 4), which are important for receptor dimerization. In the absence of a ligand, HER receptors normally exist as inactive monomers, known as the "tied" structure, which is characterized by the close interaction of domains 2 and 4. Ligand binding to the extracellular domain initiates a conformational rearrangement, exposing dimerization domains 2 and 4. Therefore, the binding of growth factors to HER receptors induces conformational changes that allow dimerization of the receptor. After dimerization of the extracellular receptor, the transmembrane helices change to an active conformation that allows the domains of the intracellular kinase to transautophosphoryle.

This phosphorylation event allows for the recruitment of specific downstream signaling proteins.

[0004] Epidermal growth factor receptor 1 (EGFR) has been causally implicated in malignancy in humans. In particular, increased EGFR expression has been observed in breast, bladder, lung, head, neck and stomach cancers, as well as in glioblastomas.

[0005] Human epidermal growth factor receptor 2 (HER2, also known as ErbB2 or Neu; UniProtKB/Swiss-Prot No. PO4626) consists of 1233 amino acids and is structurally similar to EGFR, with an extracellular domain consisting of four subdomains 1-4, a transmembrane domain, a juxtamembrane domain, an intracellular cytoplasmic tyrosine kinase, and a regulatory C-terminal region. However, the structure of the extracellular region of HER? it is different in important respects from other EGF receptors. In the other EGF receptors, in an unactivated state, domain 2 binds to domain 4. After binding to domains 1 and 3, the activating growth factor (ligand) selects and stabilizes a conformation that allows a dimerization arm extends from domain 2 to interact with an ErbB dimer pair. HER2, on the other hand, has a fixed conformation that resembles the ligand-activated state of the other members of the receptor: the domain 2-4 interaction is absent and the dimerization loop in domain 2 is continuously exposed. HER2 is activated through the formation of heterodimeric complexes with other members of the ErbB family and thus indirectly regulated by EGFR and HER3 ligands. HER2 is the preferred heterodimerization partner of the three other ErbB receptors, increasing the affinity of the other ErbB receptors for their ligands by reducing the dissociation rate of the ligand-receptor complex, whereby HER2 increases and prolongs signaling.

[0006] An excess of HER2 on the cell surface causes the transformation of epithelial cells of various tissues. Amplification of the human homolog of the neu gene (also known as HER2) is seen in breast and ovarian cancers, and correlates with poor prognosis (US 4,968,603). HER2 overexpression has also been observed in other carcinomas, including carcinomas of the stomach, endometrium, salivary gland, lung, kidney, colon, thyroid, pancreas, and bladder. Antibodies targeting HER2

[0007] Drebin and colleagues raised antibodies against the mouse neu gene product, p185<neu> described in US 6,733,752.

[0008] Hudziak et al. (1989, Mol. Cell. Biol. 9(3), 1165-1172) describe the generation of a panel of HER2 antibodies that have been characterized using the human breast tumor cell line SKBr-

3. Using a cell proliferation assay, maximal inhibition was achieved with an antibody called 4D5. It was also found that the 4D5 antibody sensitizes breast tumor cell lines with HER?2 overexpression to the cytotoxic effects of TNF-[alpha]; see also US 5,677,171. A recombinant humanized version of the 4D5 murine HER2 antibody (huMAb4D5-8, rhuMAb HER2, trastuzumab, or HERCEPTIN; US 5,821,337) is clinically active in patients with HER2 overexpression metastatic breast cancer who have previously received extensive anticancer therapy. Herceptin is approved in combination with chemotherapy for use in patients with HER2-positive metastatic stomach (gastric) cancer.

[0009] Herceptin is widely used in the treatment of patients with both early and metastatic breast cancer, whose tumors overexpress the HER2 protein and/or have HER2 gene amplification. Treatment of breast cancer patients with Herceptin/trastuzumab is, for example, recommended and now routine for patients with HER2 positive disease; see US 2002/0064785 , US 2003/0170234A1 , US2003/0134344 and US 2003/0147884 . The prior art therefore focuses on the eligibility of breast cancer patients for trastuzumab/Herceptin therapy based on a high level of HER?2 protein expression (eg, defined as HER2(3+) by immunohistochemistry (IHC)). HER2 positive disease in breast cancer is defined as present if a high level of HER2 (protein) expression is detected by immunohistochemical methods (eg HER2(+++) or as amplification of the HER2 gene (eg , a number of copies of the HER2 gene greater than 4 copies of the HER2 gene per tumor cell) or both), found in samples obtained from patients, such as breast tissue biopsies or breast tissue resections or in tissue derived from sites metastatic. A frequently applied method to detect HER2 overexpression and amplification at the gene level is fluorescence in situ hybridization (FISH), which is also described in US 2003/0152987.

[0010] Pertuzumab, a humanized antibody, is the first of a new class of agents known as HER dimerization inhibitors (HDIs). Pertuzumab binds to HER2 in its dimerization domain, thereby inhibiting its ability to form active heterodimer receptor complexes, thereby blocking the downstream signal cascade that ultimately results in cell growth and division . Pertuzumab is targeted against extracellular domain 2 of HER2. Unlike trastuzumab, which acts by binding to domain 4 of HER2, pertuzumab is an inhibitor of HER dimerization that inhibits the dimerization of HER2 with HER3 and the other members of the EGFR receptor family in the presence of their respective ligands activators. By blocking complex formation, pertuzumab prevents growth-stimulating effects and cell survival signals activated by HER1, HER3 and HER4 ligands. Pertuzumab has been approved by the FDA under the name Perjeta for treatment in combination with trastuzumab and docetaxel for patients with HER2-positive metastatic breast cancer who have not received prior anti-HER2 therapy or chemotherapy for the disease metastatic. Pertuzumab is a fully humanized recombinant monoclonal antibody based on the framework sequences of human IgG1 ([kappa]). Patent publications relating to pertuzumab and patient selection for therapy with it include: US20060073143A1; US20030086924; US20040013667A1 and US20040106161A1.

[0011] For trastuzumab, although known to have clinical benefits in terms of, for example, prolonged survival in combination with chemotherapy compared to chemotherapy alone, the majority of patients with HER?2 positive breast cancer were non-responders (rate of 45% overall response for Herceptin + chemotherapy vs. 29% for chemotherapy alone).

[0012] Thus, although monoclonal antibody therapy directed against HER2 has been shown to provide an improved treatment, for example, in metastatic breast cancer that overexpresses HER2, there is still considerable room for improvement. Non-antibody scaffolds targeting HER2

[0013] Recently, alternative targeting proteins have been proposed, which are more diverse in molecular structure than antibody fragments derived from human immunoglobulins and antibody-derived constructs and formats, thus allowing for additional molecular formats when creating heterodimeric sets and multimeric, leading to new biological functions. Several of these targeting proteins have been described (reviewed in (Binz et al, 2005, Nat. Biotech, 23, 1257-1268)). Non-limiting examples of such targeting proteins are camelid antibodies, protein scaffolds derived from protein A domains (termed "Affi-bodies", Affibody AB), tendamistat (an alpha-amylase inhibitor, a 74 beta sheet protein amino acids from Streptomyces tendae), fibronectin, lipocalin ("Anticalins", Pieris), T cell receptors, ankyrins (designated ankyrin repeat proteins termed "DARPins", Univ. Zurich and Molecular Partners; see US20120142611), A domains of various receptors ("Avimers", Avidia) and PDZ domains, fibronectin domains (FN3) ("Adnectins", Adnexus), consensus fibronectin domains ("Centyrins", Centyrex/Johnson & Johnson) and Ubiquitin ("Affilins", SCIL Proteins) and notins (Moore and Cochrane, 2012, Methods in Enzymology 503, 223-251 and references cited therein).

[0014] From these proteins, multimeric and multispecific sets can be constructed (Caravella and Lugovskoy, 2010, Current Opinions in Chemical Biology, 14, 520-528; Vanlandschoot et al., 2011, Antiviral Research 92, 389 -407; Lófblom et al., 2011, Current Opinion in Biotechnology, 22, 843-848, Boersma et al., 2011, Curr. Opin. Biotechnol., 22, 849-857). It is also possible to fuse these and other peptide domains to antibodies to create so-called Zybodies (Zygenia Inc., Gaithersburg, MD).

[0015] All of these scaffolds, with different inherent properties, have in common that they can be targeted to link specific epitopes, using selection technologies well known to professionals in the field (Binz et al., Ibid.).

[0016] For example, the different individual domains of HER2 can be expressed individually in insect cells, using a baculovirus expression system, as demonstrated for domain 1 and domain 4 (Frei et al/., 2012, Nat Biotechnol , 30, 997-1001). In this way, it is guaranteed that the selected connectors will be

will be directed to the domain of interest.

The HER2 domains can then be biotinylated as previously described (Zahnd et al., 2006, J.

Biol.

Chem. 281 (46), 35167-75) and thus immobilized on streptavidin coated magnetic beads or on streptavidin or neutrovidin coated microtiter plates (Steiner et al., 2008, J.

Mol.

Biol. 382, 1211-1227; Zahnd et al., 2007, J.

Mol.

Biol. 369, 1015-1028.). The HER2 domains immobilized in this way can then serve as targets for various protein libraries in phage display or ribosome display format.

A wide variety of different antibody libraries have been published (Mondon P. et a/., 2008, Frontiers in Bioscience. 13, 1117-1129) and the technology of selection of binding antibodies is well known by the profession. professionals in the area.

Phage display is a suitable format for antibody fragments (Fab fragments, scFv fragments or single domain antibodies) (Hoogenboom, 2005, Nature Biotechnology., 23(9), 1105-1116) and any other scaffolds that contain disulfide bonds, but can also be used for scaffolds that do not contain disulfide bonds (eg Steiner et at., 2008, J.

Mol.

Biol., 382, 1211-1227) (Rentero et al., 2011, Chimia., 65(11), 843-5, Skerra A., 2007, Current Opinion in Biotechnology., 18(4), 295-304) . Likewise, ribosome display can be used for antibody fragments (Hanes et a/l., 2000, Nat.

Biotechnol., 18, 1287-1292) and for other anjdaimes (Zahnd et al., 2007, Nat.

Methods, 4, 269-279; Zahnd et al., 2007, J.

Mol.

Biol., 369, 1015-28.). A powerful third technology is yeast display (Pepper et al., 2008, Combinatorial Chemistry & High Throughput Screening, 11(2), 127-134). In this case, a library of the binding protein of interest is displayed on the yeast surface, and the respective domain of HER2 is directly labeled with a fluorescent dye or its his-tag is detected with an anti-histag antibody which, in turn, is detected with a secondary antibody. Such methods are well known to professionals in the field (Boder et al., 2000, Methods in Enzymology, 328, 430-44).

[0017] Another engineering possibility represents the connection of these connectors to create superior bispecific or multivalent binding molecules. Such a connection can be achieved genetically by fusions of two or more of these binding molecules, or chemically by crosslinking separately expressed molecules, or by adding a dimerization domain, including separate dependent claims for each or any combination thereof ( see, for example, Stefan et al., 2011, J. Mol. Biol., 413, 826-843; Boersma et al., 2011, J. Biol. Chem., 286, 41273-41285).

[0018] A camelid anti-H[ER2 bispecific antibody construct (Bispecific Nanobody) is shown in the US2011 document

0059090. The document refers to a bispecific molecule that simultaneously targets HER2 in extracellular domain 2, defined by competition with pertuzumab, and in domain 4, defined by competition with trastuzumab. This molecule was described as exhibiting stronger antiproliferative activity than trastuzumab (Herceptin) in a direct comparison in an in vitro cell culture model using the SKBr3 cell line.

[0019] Due to the absence of any known HER2 specific ligand, current HER2 targeting strategies aim to block receptor dimerization by binding to the interaction interface. Current knowledge of HER2 receptor dimerization is based primarily on the crystal structure of the ligand-bound form of the EGFR homodimer, which is widely accepted as the active mode of all members of the EGF receptor family (Garret et al., 2002, Cell, 110, 763-773). The two EGFR molecules have a “back-to-back” interaction. Extending these discoveries to

HER? and its possible interaction with other members of the EGFR family, an interaction interface is present in domain 2 of the extracellular part of HER2. Pertuzumab binds to domain 2 and is known to block receptor interaction at this interface. Another known interaction is present in domain 4 of the extracellular part of HER2. This interaction interface is presumably blocked by trastuzumab. However, both antibodies, trastuzumab and pertuzumab, even when applied simultaneously, are not able to completely block all HER2 interactions. It is believed that the interaction of the extracellular part and the kinase domain of HER2 is linked in such a way as to allow some residual interactions even in the blocked state by trastuzumab and pertuzumab, which is in agreement with the crystal structure data (Lu et a/. , 2010, Mol. Cell. Biol., 22, 5432-5443). The bispecific ligand mentioned above that binds to both epitopes (pertuzumab and trastuzumab) simultaneously (US 2011/0059090) reduces cell growth in a cell culture model by approx. 50%, compared to a reduction of about 40% achieved by trastuzumab. This same effect, however, can also be obtained by treatment with a mixture of trastuzumab and pertuzumab.

[0020] Patent application WO 2014/060365 describes bispecific HER2 targeting agents comprising a first polypeptide linker that binds to extracellular domain 1 of HER2 (epitope D1) and a second polypeptide linker that binds to the domain extracellular 4 of HER2 (epitope D4), in which the first and second polypeptide linker are separated by the linker. The most active of these biparatopic binding agents predominantly bind to two separate HER2 molecules in an intermolecular binding mode. Thus, these biparatopic bivalent linkers cross-link HER2 ECD1 from one HER2 and HER2 ECDA4 from the other molecule

HER?2 through these paratopes and a preferably short peptide ligand, which favors intermolecular over intramolecular binding.

The bivalent binding mode of bi-paratopic binding agents will induce a polymerization of HER2 receptors on the cell surface which, consequently, are not able to form productive HER2Q/HER3 or HER2/EGFR heterodimers or HER2/HER2 homodimers (Tamaskovic et al., 2016, Jost et al., 2013). Consequently, this leads to an inhibition of HER2 and HER3 phosphorylation in HER2 overexpressing cancer cells, which subsequently leads to cessation of proliferative and anti-apoptotic HER2/HER3 receptor signaling and ultimately induction of cell death by apoptosis.

However, these biparatopic binding agents do not affect total HER?2 receptor expression levels (Tamaskovic et al., 2016). The total expression of the HER2 receptor remains comparatively high, which can cause an incomplete inactivation of the HER2 receptors. Furthermore, these constructs do not have the desired pharmacokinetic properties, which are necessary for systemic applications.

Desired functions, such as long serum half-life in the bloodstream or mechanisms of protein recycling through binding to the FcRn receptor, are not implemented in these constructs.

Furthermore, these biparatopic constructs do not take advantage of antibody effector functions such as complement-dependent cytotoxicity (CDC) or antibody-dependent cell-mediated cytotoxicity (ADCC). These effector functions may be beneficial to further enhance the antitumor activity of biparatopic binding agents in vivo.

Finally, these biparatopic binding agents are potentially likely to induce an immune response because they were not additionally designed to avoid T cell epitopes.

This can lead to a significant reduction in tolerability and serum level at repeated doses in immunocompetent patients.

In addition, a biparatopic HER2-targeting tetravalent drug-antibody conjugate has been described which comprises a fusion of Trastuzumab and 39S antibody variable sequences binding to the D4 and D2 epitopes of HER?2 (Li et al., 2016, Cancer Cell, 29, 117-129). This conjugate has been marketed under the name MEDI4276 by Medimmune and has been tested in a phase 1/2 clinical trial (NOT02576548). However, this construct of biparatopic IgG fusion in its armless form, that is, without the fusion of the toxic load of Tubulisin, is not sufficient to induce the inhibition of cancer cell proliferation.

In contrast, the unconjugated version of this IgG fusion molecule induces activation of cancer cell proliferation at high concentrations in cancer cell models overexpressing HER?2. This may be caused by binding to HER2 ECD2 and HER2 ECDA4 in a way that enhances the formation of signaling competent HER2 homo- and heterodimers.

On the other hand, the unconjugated IgG fusion protein version of this tetravalent biparatopic HER?2 targeting antibody can induce downregulation of HER2 receptor expression (Li et a/., 2016, Cancer Cell, 29, 117- 129). In summary, the tetravalent scFv 4D5-IgG 398 fusion protein downregulates HER2 expression, but does not show inhibition of HER2 signaling and instead leads to activation of cancer cell proliferation in specific models of overexpression of HER?2. It also shows that HER2 downregulation and cancer cell growth inhibition are simply not linked.

Clearly, inhibition of the HER2-dependent signaling pathways of a HER?2 targeting agent would be essential for clinical applications.

For example, trastuzumab (Herceptin) blocks HER3 signaling and shows a significant reduction in cell proliferation in HER2 overexpressing cancers that do not have a PISK pathway activating mutation.

[0022] Based on the above-mentioned state-of-the-art technique, the aim of the present invention is to provide means and methods for providing a targeting agent to the HER2 that is improved in relation to the aforementioned disadvantages of the prior art, in particular to provide a HER2 targeting agent exhibiting improved inhibition of HER2 signaling, downregulation of expression, polymerization and assembly, inhibition of receptor diffusion, degradation and/or inhibition of recycling, in the absence of additional small molecule toxins linked to the targeting agent to HER2. This objective is achieved by the subject of the independent claims of this descriptive report. Invention Summary

[0023] The invention relates to a tetrameric polypeptide comprising or consisting of

[0024] a first polypeptide chain comprising, in the N to C orientation, a first VL antigen-binding domain and a first constant domain CL,

[0025] a second polypeptide chain comprising, in the N to C orientation, a first VH antigen-binding domain, a first CH1 constant domain, a first CH2 constant domain, and a first CH3 constant domain,

[0026] a first linker that specifically binds to a D4 epitope of HER2, wherein the first linker is comprised of the first polypeptide chain and linked to the N-terminus of the first VL antigen-binding domain by a first linker of interdomain amino acid, or the first linker is comprised of the second polypeptide chain and linked to the N-terminus of the first VH antigen-binding domain by a first interdomain amino acid linker,

[0027] in which the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain, together, constitute a second linker, particularly a Fab domain, which binds specifically to a D1 epitope of HER?2,

[0028] a third polypeptide chain comprising, in the N to C orientation, a second VL antigen-binding domain and a second constant CL domain,

[0029] a fourth polypeptide chain comprising, in the N to C orientation, a second VH antigen-binding domain, a second CH1 constant domain, a second CH2 constant domain, and a second CH3 constant domain.

[0030] a third linker that specifically binds to a D4 epitope of HER2, wherein the third linker is comprised of the third polypeptide chain and linked to the N-terminus of the second VL antigen-binding domain by a second linker of interdomain amino acid, or the third linker is comprised of the fourth polypeptide chain and linked to the N-terminus of the second VH antigen-binding domain by a second interdomain amino acid linker,

[0031] in which the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together constitute a fourth ligand, particularly a Fab domain, which specifically binds to a D1 epitope of HER?2,

[0032] The tetrameric polypeptide according to the invention, therefore, comprises twice two different HER2 binding paratopes, in other words, it is tetravalent. The polypeptide is biparatopic because it contains binding sites to two distinct HER?2 epitopes, namely, D1 and D4 on a single molecule.

[0033] Surprisingly, the polypeptide exhibits inactivation of

superior HER?2 compared to conventional antibodies and divalent biparatopic polypeptides (composed of a total of two binding paratopes) and has additional effects on cell growth and proliferation, apoptosis and other forms of cell death, internalization of HER2 and HER?2 recycling inhibition, downregulation of HER2 expression and HER2 degradation, HER?2 crosslinking, inhibition of HER2 dimerization, and decreased HER2 receptor surface mobility. Furthermore, the increased molecular size excludes renal filtration and FcRn-mediated recycling will increase the pharmacokinetic properties. Finally, the presence of the Fc part also allows antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC) to occur. Finally, all sequences are from human antibodies and therefore the construct consists of non-immunogenic sequences. In summary, the tetrameric polypeptides, according to the present invention, are promising candidates for systemic cancer therapy that expresses HER?2.

[0034] In another embodiment, the present invention relates to a pharmaceutical composition comprising at least one of the compounds of the present invention or a pharmaceutically acceptable salt thereof and at least one pharmaceutically acceptable carrier, diluent or excipient.

[0035] The invention also relates to tetrameric polypeptide for use in a method for preventing or treating a malignant neoplastic disease, an isolated nucleic acid encoding the polypeptide, a host cell for producing the polypeptide, and a method for get the polypeptide. Detailed description of the invention Terms and definitions

[0036] Unless otherwise defined, all technical terms

The unique and scientific meanings used herein have the same meaning commonly understood by one of ordinary skill in the art (eg, in cell culture, molecular genetics, nucleic acid chemistry, hybridization techniques, and biochemistry). Standard techniques are used for molecular, genetic, and biochemical methods (see, in general, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Ed, John Wiley & Sons, Inc.) and chemical methods.

[0037] The term "an individual comprises an object", in the context of this descriptive report, includes discrete modalities in which the individual consists of the object, in other words, where the term understand is synonymous with "consist of". In other discrete modalities, the object is one of several different ones comprised in the object.

[0038] The term linker, in the context of this descriptive report, refers to a region of a polypeptide that binds to a target, particularly HER?2.

[0039] The term interdomain amino acid linker, in the context of this specification, refers to a polypeptide linker that covalently connects the C-terminus of a first polypeptide domain to the N-terminus of a second polypeptide domain.

[0040] The term epitope, in the context of this descriptive report, refers to a region of an antigen molecule to which an antibody binds.

[0041] The term Fab domain in the context of this specification refers to an antibody molecule comprising a first chain consisting of a C-terminal VL domain (covalently) linked to a CL domain and a second chain consisting of a C-terminally linked VH domain (covalently) to a CH1 domain, wherein the CL domain and CH1 domain are linked by a disulfide bond.

[0042] The term VL antigen-binding domain, in the context of this specification, refers to the variable domain of the light chain of an antibody, particularly of an immunoglobulin G light chain.

[0043] The term VH antigen binding domain, in the context of this specification, refers to the variable domain of the heavy chain of an antibody, particularly of an immunoglobulin G heavy chain.

[0044] The term constant domain CL, in the context of this specification, refers to the constant domain of the light chain of an antibody, particularly of an immunoglobulin G light chain.

The term CH1 constant domain, in the context of this specification, refers to the first constant domain of the heavy chain of an antibody, particularly of an immunoglobulin G heavy chain.

The term CH2 constant domain, in the context of this specification, refers to the second constant domain of an antibody heavy chain, particularly an immunoglobulin G heavy chain.

The term CH3 constant domain, in the context of this specification, refers to the third constant domain of an antibody heavy chain, particularly an immunoglobulin G heavy chain.

[0048] The term single-chain variable fragment, in the context of this specification, refers to a VH antigen-binding domain (also called scFv heavy chain) covalently linked to a VL antigen-binding domain (also called scFv light chain) by a polypeptide linker ( |i- chain

of scFv).

[0049] In this descriptive report, the term positive, when used in the context of expression of a marker, refers to the expression of an antigen tested by an antibody labeled with fluorescence, in which the fluorescence of the marker in the structure ( for example, a cell) referred to as “positive” is at least 30% larger (> 30%), particularly 250% or 280%, in median fluorescence intensity when compared to staining with an antibody labeled with fluorescence of isotype-compatible that does not specifically bind to the same target. Such a marker expression is indicated by a superscript (*) after the marker name, eg CD4*.

[0050] In this descriptive report, the term negative, when used in the context of expression of a marker, refers to the expression of an antigen tested by an antibody labeled with fluorescence, in which the median fluorescence intensity is less 30% higher, particularly less than 15% higher, than the median fluorescence intensity of an isotype-matched antibody that does not specifically bind to the same target. Such a marker expression is indicated by a minus superscript () after the marker name, eg CD127-.

[0051] High expression of a marker, eg high expression of CD25, refers to the expression level of such a marker in a clearly distinguishable cell population that is detected by FACS, showing the highest fluorescence intensity per cell in compared to other populations characterized by a lower fluorescence intensity per cell. A high expression is indicated by the superscript "high" or "hi" after the marker name, for example, CD25º'º. The term “highly expressed” refers to the same characteristic.

[0052] Low expression of a marker, eg low expression of CD25, refers to the expression level of such a marker in a clearly distinguishable cell population that is detected by FACS showing the lowest fluorescence intensity per cell compared to with the other populations characterized by a higher fluorescence intensity per cell. A low expression is indicated by the superscript "low" or "lo" after the marker name, eg CD25”*x2. The term “is lowly expressed” refers to the same characteristic.

[0053] The expression of a marker can be tested using techniques such as fluorescence microscopy, flow cytometry, ELIS-POT, ELISA or multiplex analysis.

[0054] The term polypeptide, in the context of this specification, refers to a molecule consisting of 50 or more amino acids that form a linear chain in which the amino acids are connected by peptide bonds. The amino acid sequence of a polypeptide can represent the amino acid sequence of an entire protein (as found physiologically) or its fragments. The terms "polypeptides" and "protein" are used interchangeably herein and include proteins and fragments thereof. Polypeptides are described herein as amino acid residue sequences.

[0055] The term peptide, in the context of this descriptive report, refers to a molecule consisting of up to 50 amino acids, in particular 8 to 30 amino acids, more particularly 8 to 15 amino acids, which form a linear chain in that amino acids are connected by peptide bonds.

The amino acid residue sequences are provided from the amino terminus to the carboxyl terminus. Capital letters for sequence positions refer to L-amino acids in the one-letter code.

(Stryer, Biochemistry, 3rd ed. p. 21). Lowercase letters for amino acid sequence positions refer to the corresponding D- or (2R)-amino acids. Sequences are written from left to right in the direction of the amino terminus for the carboxy. According to standard nomenclature, amino acid residue sequences are denoted by a three-letter or single-letter code as indicated below: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N) , Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (Gln, Q), Glutamic Acid (Glu, E), Glycine (Gly, G), Histidine (His, H), Isoleucine (Ile , 1), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine ( Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y) and Valine (Val, V). J is leucine or isoleucine.

[0057] The term gene refers to a polynucleotide containing at least one open reading frame (ORF) that is capable of encoding a particular polypeptide or protein after being transcribed and translated. A polynucleotide sequence can be used to identify larger fragments or full-length coding sequences of the gene with which they are associated. Methods of isolating longer fragment sequences are known to those of skill in the art.

[0058] The terms gene expression or, alternatively, gene product refer to the processes - and products thereof - of nucleic acids (RNA) or amino acids (eg, peptide or polypeptide) being generated when a gene is transcribed and translated .

As used herein, expression refers to the process by which DNA is transcribed into mRNA and/or the process by which the transcribed mMRNA is subsequently translated into peptides, polypeptides or proteins. If the polynucleotide is derived from genomic DNA, expression may include splicing the mRNA in an eu-

karyotic. Expression can be assayed at the level of transcription and translation, in other words, MRNA and/or protein product.

Sequences similar or homologous (e.g., at least about 70% sequence identity) to the sequences described herein also form part of the invention. In some embodiments, the sequence identity at the amino acid level can be about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99 % or superior. At the nucleic acid level, the sequence identity can be about 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher. Alternatively, there is substantial identity when the nucleic acid segments will hybridize under selective hybridization conditions (e.g., very high stringency hybridization conditions), to strand complement. Nucleic acids can be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0061] Calculations of "homology" or "sequence identity" or "similarity" between two sequences (the terms are used interchangeably here) are performed as follows. Sequences are aligned for optimal comparison purposes (eg gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . In a particular embodiment, the length of an aligned reference sequence for comparison purposes is at least 30%, particularly at least 40%, more particularly at least 50%, even more particularly at least 60% and even more particularly at least 70%, 80%, 90%, 100% of the length of the reference sequence. Then, the amino acid or nucleotide residues at the corresponding amino acid positions or nucleotide positions are compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "homology" is equivalent to an amino acid or nucleic acid "identity"). The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In the case of circularly related proteins, the sequence from one of the partners needs to be properly split and aligned into two sections to achieve optimal alignment of the functionally equivalent residues needed to calculate percent identity.

[0062] In the context of this specification, the terms sequence identity and percentage of sequence identity refer to the values determined by comparing two aligned sequences. Sequence alignment methods for comparison are well known in the art. Alignment of sequences for comparison can be performed by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math., 2, 482, by the global alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol., 48, 443, by the similarity search method of Pearson and Lipman, 1988, Proc. Nat. Sci., 85, 2444 or by computerized implementations of these algorithms, including, but not limited to: CLUSTAL, GAP, BESTFIT, BLAST, FASTA and TFASTA. Software for performing BLAST analysis is publicly available, for example, through the National Center for Biotechnology-Information (http://blast.ncbi.nlm.nih.gov/).

[0063] An example for comparing amino acid sequences.

dos is the BLASTP algorithm that uses the default settings: Expected Threshold: 10; Word size: 3; Maximum pairings in a query interval: 0; Matrix: BLOSUM62; Gap costs: Existence 11, Extent 1; Compositional Adjustments: Conditional adjustment of the compositional scoring matrix. An example for comparing nucleic acid sequences is the BLASTN algorithm, which uses the default settings: Expected Threshold: 10; Word Size: 28; Maximum pairings in a query interval: 0; Matching/mismatching scores: 1.-2; Gap costs: Linear. Unless otherwise indicated, sequence identity values provided herein refer to the value obtained using the BLAST software suite (Altschul et al., 1990, J. Mol. Biol., 215, 403-410 ) using the standard parameters identified above for protein and nucleic acid comparison, respectively.

[0064] In the context of this specification, the term antibody refers to whole antibodies including, but not limited to, immunoglobulin type G (IgG), type A (IgA), type D (I9D), type E (IgE) or type M (IgM), any antigen-binding fragment or single chains thereof and related constructs or derivatives. A complete antibody is a glycoprotein comprising at least two heavy (H) and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (Vx) and a heavy chain constant region (Cr). The heavy chain constant region is composed of three domains, ChH1, ChH2 and Cx3. Each light chain is composed of a light chain variable region (herein abbreviated as V.) and a light chain constant region (C1). The light chain constant region is composed of a domain, Cr. The variable regions of heavy and light chains contain a binding domain that interacts with an antigen. Antibody constant regions can mediate immunoglobulin binding to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component of the classical complement system. Likewise, the term encompasses a so-called nanobody or single-domain antibody, an antibody fragment that consists of a single variable monomeric antibody domain.

[0065] In the context of this specification, the term humanized antibody refers to an antibody originally produced by immune cells of a non-human species, whose protein sequences have been modified to increase its similarity with variants of naturally produced antibodies in humans. The term humanized antibody as used herein includes antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Additional framework region modifications can be made to the human framework sequences as well as to the CDR sequences derived from the germline of another mammalian species.

[0066] The term antibody-like molecule, in the context of this specification, refers to a molecule capable of specific binding to another molecule or target with high affinity / a Kd < 10E-8 mol/l. An antibody-like molecule binds to its target in a manner similar to the specific binding of an antibody. The term antibody-like molecule encompasses a repeat protein, such as a engineered ankyrin repeat protein (Molecular Partners, Zurich), a engineered antibody mimetic protein exhibiting high-affinity and highly specific target protein binding (see US 2012/142611, US 2016/250341, US 2016/075767 and US 2015/368302, all of which are incorporated herein by reference). The term antibody-like molecule additionally includes, but is not limited to, a polypeptide derived from armadillo repeat proteins, a polypeptide derived from leucine-rich repeat proteins, and a polypeptide derived from tetratricopeptide repeat proteins.

The term antibody-like molecule further encompasses a specific binding polypeptide derived from a protein A domain, fibronectin domain FN3, consensus fibronectin domains, a lipocalin (see Skerra, 2000, Biochim. Biophys. Acta, 1482(1-2), 337-50), a polypeptide derived from a zinc finger protein (see Kwan et al., 2003, Structure, 11(7), 803-813), Src homology domain 2 (SH2) or Src homology domain 3 (SH3), a PDZ domain, gamma-crystalline, ubiquitin, a cysteine-knot polypeptide, or a notin, cystatin, Sac7d, a triple helix spiral coil (also known as alfabodies), a Kunitz domain or a Kunitz-type protease inhibitor and a 32-2 carbohydrate-binding module.

[0068] The term polypeptide derived from protein A domains refers to a molecule that is a derivative of protein A and is capable of specifically binding to the Fc region and the Fab region of immunoglobulins.

[0069] The term armadillo repeat protein refers to a polypeptide comprising at least one armadillo repeat, wherein an armadillo repeat has characteristics of a pair of alpha helices that form a staple-like structure.

[0070] The term humanized camelid antibody, in the context of this specification, refers to an antibody that consists only of the heavy chain or the variable domain of the heavy chain (VHH domain) and whose amino acid sequence has been modified to increase its similarity with antibodies naturally produced in humans and therefore exhibit reduced immunogenicity when administered to a human. A general strategy for humanizing camelid antibodies is shown in Vincke et al. 2009, J Biol Chem., 284(5), 3273-3284, and US 2011/165621.

[0071] In the context of this descriptive report, the term crystallizable fragment region (Fc) is used in its known meaning in the art of cell biology and immunology; refers to a fraction of an antibody comprising two identical heavy chain fragments composed of a Cx2 and a Cr3 domain, covalently linked by disulfide bonds.

[0072] The term specific binding, in the context of this descriptive report, refers to a property of ligands that bind to their target with a certain affinity and specificity to the target. The affinity of such a linker is indicated by the dissociation constant of the linker. A specifically reactive ligand has a dissociation constant of < 10 "mol/L when binding to its target, but a dissociation constant at least three orders of magnitude greater in its interaction with a molecule with a chemical composition globally similar to the target, but a different three-dimensional structure.

[0073] In the context of this descriptive report, the term dissociation constant (KD) is used in its known meaning in the art of chemistry and physics; refers to an equilibrium constant that measures the propensity of a complex composed of [mainly two] different components to reversibly dissociate into its constituent components. The complex can be, for example, an antibody-antigen ADbAg complex composed of antibody Ab and antigen Ag. Kp is expressed in molar concentration [mol/]] and corresponds to the concentration of [Ab] in which half of the sites of binding of [Ag] are occupied, in other words, the concentration of unbound [Ab] is equal to the concentration of complex [AbAg]. The dissociation constant can be calculated according to the following formula: 12 BIA lAbAg]

[0074] [Ab]: antibody concentration; [Ag]: antigen concentration; [ADAg]: antibody-antigen complex concentration

[0075] In the context of this descriptive report, the terms dissociation rate (Koff;[1/s]) and association rate (Kon; [1/s*M]) are used in their known meaning in the art of chemistry and of physics; they refer to a rate constant that measures the dissociation (Koff) or association (Kon) of an antibody with its target antigen.

[0076] Koff and Kon can be determined experimentally using methods well established in the art. One method to determine the Koff and Kon of an antibody uses surface plasmon resonance. This is the principle behind biosensor systems such as the Biacore& or ProteOnêO system. They can also be used to determine the dissociation constant KD using the following formula: Ko]

[0077] As used herein, the term pharmaceutical composition refers to a compound of the invention, or a pharmaceutically acceptable salt.

the same, together with at least one pharmaceutically acceptable carrier. In certain embodiments, the pharmaceutical composition according to the invention is provided in a form suitable for topical, parenteral or injectable administration.

[0078] As used herein, the term pharmaceutically acceptable carrier includes any solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, binders, excipients, disintegrating agents, lubricants, sweetening agents, flavoring agents, colorants and the like and combinations thereof, as would be known to those skilled in the art (see, for example, Remington: the Science and Practice of Pharmacy, ISBN 0857110624).

[0079] As used herein, the term treating or treating any disease or disorder (for example, cancer) refers, in one modality, to ameliorating the disease or disorder (for example, delaying or interrupting or reducing the development of the disease or at least one of its clinical symptoms). In another embodiment, "treating" or "treatment" refers to alleviating or ameliorating at least one physical parameter, including those that may not be discernible by the patient. In yet another embodiment, "treating" or "treatment" refers to modulating the disease or disorder, physically (eg, stabilization of a discernible symptom), physiologically (eg, stabilization of a physical parameter), or both. Methods for evaluating the treatment and/or prevention of diseases are generally known in the art, unless specifically described below.

[0080] In the context of this descriptive report, the term dimer refers to a unit consisting of two subunits.

[0081] In the context of this descriptive report, the term homo-

dimer refers to a dimer composed of two subunits that are identical or are highly similar members of the same class of subunits.

[0082] In the context of this specification, the term amino acid linker refers to a polypeptide of variable length that is used to connect two polypeptides to generate a single-chain polypeptide. Exemplary embodiments of linkers useful for the practice of the invention specified herein are oligopeptide chains consisting of 1, 2, 3, 4, 5, 10, 20, 30, 40 or 50 amino acids. A non-limiting example of an amino acid linker is the polypeptide GGGGSGGGGS (SEQ ID NO 83).

[0083] A first aspect of the invention relates to a tetrameric polypeptide.

[0084] According to a first alternative of the first aspect, the tetrameric polypeptide comprises or consists of

[0085] a first polypeptide chain comprising, in the N to C orientation, a first VL antigen-binding domain and a first constant CL domain,

[0086] a second polypeptide chain comprising, in the N to C orientation, a first VH antigen-binding domain, a first CH1 constant domain, a first CH2 constant domain, and a first CH3 constant domain,

[0087] a first linker that specifically binds to a D4 epitope of HER2, wherein the first linker is comprised in the first polypeptide chain and linked to the N-terminus of the first VL antigen-binding domain by a first linker of interdomain amino acid, or the first linker is comprised of the second polypeptide chain and linked to the N-terminus of the first VH antigen-binding domain by a first interdomain amino acid linker,

[0088] in which the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain, together, constitute a second linker, particularly a Fab domain, which binds specifically to a D1 epitope of HER?2,

[0089] a third polypeptide chain comprising, in the N to C orientation, a second VL antigen-binding domain and a second constant CL domain,

[0090] a fourth polypeptide chain comprising, in the N to C orientation, a second VH antigen-binding domain, a second CH1 constant domain, a second CH2 constant domain, and a second CH3 constant domain.

[0091] a third linker that specifically binds to a D4 epitope of HER2, wherein the third linker is comprised of the third polypeptide chain and linked to the N-terminus of the second VL antigen-binding domain by a second linker of interdomain amino acid, or the third linker is comprised of the fourth polypeptide chain and linked to the N-terminus of the second VH antigen-binding domain by a second interdomain amino acid linker,

[0092] wherein the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together constitute a fourth ligand, particularly a Fab domain, which specifically binds to a D1 epitope of HER?2,

[0093] According to a second alternative of the first aspect, the tetrameric polypeptide comprises or consists of

[0094] a first polypeptide chain comprising, in the N to C orientation, a first VL antigen-binding domain and a first constant CL domain,

[0095] a second polypeptide chain comprising, in the original,

tion of N to C, a first VH antigen-binding domain and a first constant domain CH1, in which particularly the second polypeptide chain further comprises a first constant domain CH2, in which more particularly the first constant domain CH2 is truncated at its C-terminal,

[0096] a first linker that specifically binds to a D4 epitope of HER2, wherein the first linker is comprised in the first polypeptide chain and linked to the N-terminus of the first VL antigen-binding domain by a first linker of interdomain amino acid, or the first linker is comprised of the second polypeptide chain and linked to the N-terminus of the first VH antigen-binding domain by a first interdomain amino acid linker,

[0097] in which the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain, together, constitute a second ligand, particularly a Fab domain, which binds specifically to a D1 epitope of HER?2,

[0098] a third polypeptide chain comprising, in the N to C orientation, a second VL antigen-binding domain and a second constant CL domain,

[0099] a fourth polypeptide chain comprising, in the N to C orientation, a second VH antigen-binding domain and a second constant domain CH1, wherein particularly the fourth polypeptide chain additionally comprises a second constant domain CH2, in which more particularly the second constant domain CH2 is truncated at its C-terminus,

[00100] a third linker that specifically binds to a D4 epitope of HER2, wherein the third linker is comprised of the third polypeptide chain and linked to the N-terminus of the second VL antigen-binding domain by a second linker of interdomain amino acid, or the third linker is comprised of the fourth polypeptide chain and linked to the N-terminus of the second VH antigen-binding domain by a second interdomain amino acid linker,

[00101] wherein the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together constitute a fourth linker, particularly a Fab domain, which specifically binds to a D1 epitope of HER?2,

[00102] All of the embodiments below can be combined with the first alternative and the second alternative of the first aspect of the invention.

[00103] The VL antigen binding domain and the CL constant domain of the first and third polypeptide chain are domains of an immunoglobulin G light chain, and the VH antigen binding domain and the CH1, CH2 and CH3 constant domains of the second and fourth polypeptide are domains of an immunoglobulin G heavy chain. In other words, the first and third polypeptide chains each comprise an immunoglobulin G light chain, and the second and fourth polypeptide chains each comprise one chain heavy immunoglobulin G. The immunoglobulin light and heavy chains of the tetrameric polypeptide according to the invention form the second and fourth linkers that specifically bind to the D1 epitope of HER?2.

Furthermore, polypeptides comprising a first and third linker that specifically bind to the D4 epitope of HER2 are fused to the N-terminus of immunoglobulin G heavy chains or immunoglobulin G light chains by an interdomain amino acid linker, resulting in a tetrameric polypeptide with four HER?2 binding sites, two of which bind to the D4 epitope and two of which bind to the D1 epitope.

[00105] — Surprisingly, the tetrameric polypeptide according to the invention exhibits superior HER2 inactivation compared to conventional antibodies and other antibody-like molecules such as divalent biparatopic polypeptides (having a total of two binding regions) and it has additional effects on cell growth, apoptosis, HER2 internalization, and HER2 degradation. Therefore, the tetrameric polypeptides, according to the present invention, are promising candidates for cancer therapy that expresses HER?2.

[00106] Without wishing to be bound by theory, it is believed that the CH1, CH2, CH3 and CL domains contribute, particularly the CH2 and CH3 domains, to the additional effects of the tetrameric polypeptides of the invention, particularly the inhibition of cell growth and proliferation, induction of apoptosis and other forms of cell death, HER2 internalization, inhibition of HER2 recycling and HER2 degradation, HER2 crosslinking, reduction of HER?2 surface mobility, downregulation of HER2 expression, inhibition of HER?2 dimerization and signaling by positioning the variable domains at a given angle and distance.

[00107] In certain embodiments, the first polypeptide chain and the third polypeptide chain comprise a sequence identity to each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95 % or more, wherein more particularly the first polypeptide chain and the third polypeptide chain are identical.

[00108] In certain embodiments, the first polypeptide chain comprises a sequence identity of 70%, 71%, 72%, 73%, 74%, 15%, 76%, 17%, 18%, 719%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% ,

98%, 99% or 100% with the third polypeptide chain.

[991909] — In certain embodiments, the second polypeptide chain and the fourth polypeptide chain comprise a sequence identity to each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, wherein more particularly the second polypeptide chain and the fourth polypeptide chain are identical.

[00110] In certain embodiments, the second polypeptide chain comprises a sequence identity of 70%, 71%, 72%, 73%, 74%, 175%, 76%, 77%, 718%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% , 98%, 99% or 100% with the fourth polypeptide chain.

In certain embodiments, the immunoglobulin light chain of the first polypeptide chain and the immunoglobulin light chain of the third polypeptide chain comprise a sequence identity to each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, wherein more particularly the immunoglobulin light chain of the first polypeptide chain and the immunoglobulin light chain of the third polypeptide chain are identical.

[00112] In certain embodiments, the immunoglobulin light chain of the first polypeptide chain comprises a sequence identity of 70%, 71%, 72%, 73%, T4%, T5%, 16%, TT%, 78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95 %, 96%, 97%, 98%, 99% or 100% with the immunoglobulin light chain of the third polypeptide chain.

In certain embodiments, the immunoglobulin heavy chain of the second polypeptide chain and the immunoglobulin heavy chain of the fourth polypeptide chain comprise a sequence identity to each other of 70% or more, particularly 80% or more , more particularly 90% or more, even more particularly 95% or more, wherein more particularly the second polypeptide chain immunoglobulin heavy chain and the fourth polypeptide chain immunoglobulin heavy chain are identical.

In certain embodiments, the immunoglobulin heavy chain of the second polypeptide chain comprises a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 17%, 18% , 19%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 294%, 95 %, 96%, 97%, 98%, 99% or 100% with the immunoglobulin heavy chain of the fourth polypeptide chain.

[00115] In certain embodiments, the first linker and the third linker comprise a sequence identity to each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, in which more particularly the first binder and the third binder are identical.

[00116] In certain embodiments, the first linker comprises a sequence identity of 70%, 71%, 72%, 73%, T4%, T5%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the third binder.

[00117] In other words, the first binder is substantially the same as the third binder.

[00118] In certain embodiments, the second linker and the fourth linker comprise a sequence identity to each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, in which more particularly the second ligand and the fourth ligand are identical.

[00119] In certain embodiments, the second linker comprises a sequence identity of 70%, 71%, 72%, 73%, T4%, T5%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%,

88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with the fourth binder.

[00120] In other words, the second binder is substantially the same as the fourth binder.

[00121] In certain embodiments, the first CH2 constant domain and the first CH3 constant domain of the second polypeptide chain interact with the second CH2 constant domain and the second CH3 constant domain of the fourth polypeptide chain, such that a tetrameric polypeptide is formed . In particular, the CH2 and CH3 constant domains of the second and fourth polypeptide chains dimerize. In particular, combined with the interaction between the first and second polypeptide chains and the third and fourth polypeptide chains, particularly through dimerization between the respective VL and VH antigen-binding domains and the constant domains CL and CH1, this results in tetramer formation of the first, second, third, and fourth polypeptides. Therefore, with respect to the formation of multimers, the tetrameric polypeptide of the present invention is similar to an antibody.

In certain embodiments, the second polypeptide chain comprises a first hinge region between the first constant domain of CH1 and the first constant domain of CH2, and the fourth polypeptide chain comprises a second hinge region between the second constant domain of CH1 and the second constant domain of CH2, wherein the first hinge region and the second hinge region mediate complex formation between the second polypeptide chain and the fourth polypeptide chain, particularly by at least one disulfide bond, more particularly by a first disulfide bond and a second disulfide bond, such that a tetrameric polypeptide is formed. The formation of complexes between the CH1 and CH2 constant domains can thus

occur through the hinge region, that is, by formation of a disulfide bond between cysteine residues, as well as in antibodies.

[00123] In certain embodiments, the CH2 constant domain and/or the CH3 constant domain of the second polypeptide chain is truncated at its C-terminus.

[00124] In certain embodiments, the CH2 constant domain and/or the CH3 constant domain of the fourth polypeptide chain is truncated at its C-terminus.

In certain embodiments, the first linker comprises or consists of a single chain variable fragment polypeptide chain comprising an scFv heavy chain, an scFv linker chain, and an scFv light chain. In certain embodiments, the scFv heavy chain is the 4D5 VH domain (particularly SEQ ID No. 80), and wherein the scFv light chain is the 4D5 VL domain (particularly SEQ ID No. 81).

In certain embodiments, the single-chain variable fragment polypeptide chain comprises, in N to C orientation, an scFv heavy chain, an scFv linker chain, and an scFv light chain.

In certain embodiments, the single-chain variable fragment polypeptide chain comprises, in N to C orientation, an scFv light chain, an scFv linker chain, and an scFv heavy chain.

[00128] In other words, the scFv heavy chain and the scFv light chain can be provided in any orientation in the polypeptide chain of the single-chain variable fragment.

In certain embodiments, the first linker comprises or consists of a single-chain variable fragment polypeptide chain comprising, in N to C orientation, a scFv heavy chain, a scFvy linker chain, and a scFv light chain .

In certain embodiments, the scFv heavy chain is the 4D5 VH domain (particularly SEQ ID No. 80), and wherein the scFv light chain is the 4D5 VL domain (particularly SEQ ID No. 81).

[00130] In certain modalities,

[00131] the scFv heavy chain of the first linker comprises or consists of a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54 and SEQ ID NO. 80, wherein more particularly the scFv heavy chain of the first linker comprises or consists of a peptide sequence identical to a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22 , SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54, and SEQ ID NO.80, and the scFv light chain of the first linker comprises or consists of a peptide sequence with a sequence identity of 70 % or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a pept sequence ide selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81, more particularly the scFv light chain of the first linker comprises or consists of a peptide sequence identical to a peptide sequence selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81.

In certain embodiments, the scFv heavy chain of the first linker comprises or consists of a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, 76 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,

99% or 100% with a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54 and SEQ ID No. 80, and the scFv light chain of the first linker comprises or consists of a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO. 26, SEQ ID No. 43 and SEQ ID No. 81.

[00133] In certain embodiments, the scFv heavy chain of the third linker comprises or consists of a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO. 54 and SEQ ID No. 80, wherein more particularly the scFv heavy chain of the third linker comprises or consists of a peptide sequence identical to a peptide sequence selected from SEQ ID No. 15, SEQ ID No. 21 , SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54 and SEQ ID NO.80, and the third linker scFv light chain comprises or consists of a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81, more particularly the scFv light chain of the third linker comprises or consists of a peptide sequence identical to a peptide sequence selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81 .

In certain embodiments, the scFv heavy chain of the third linker comprises or consists of a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16 %, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54, and SEQ ID No. 80, and the third linker scFv light chain comprises or consists of a peptide sequence with a sequence identity of 70 %, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected a from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81.

[00135] In certain embodiments, the scFv heavy chain of the first linker and the third linker each comprises or consists of a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44 , SEQ ID NO.53, SEQ ID NO.54 and SEQ ID NO.80, wherein more particularly the scFv heavy chain of the first linker and the third linker each comprises or consists of a peptide sequence identical to one peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54 and SEQ ID NO.80, and the scFv light chain of the first linker and the third linker each comprise or consist of a peptide sequence with a sequence identity of 70% or greater, p particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43, and SEQ ID No. 81, wherein more particularly the scFv light chain of the first linker and the third linker each comprises or consists of a peptide sequence identical to a selected peptide sequence a from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81.

In certain embodiments, the scFv heavy chain of the first linker and the third linker each comprises or consists of a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, 74 %, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID No.15, SEQ ID No.21, SEQ ID No. 22, SEQ ID No. 23, SEQ ID No. 44, SEQ ID No. 53, SEQ ID No. 54 and SEQ ID No. 80, and the scFv light chain of the first linker and the third linker each comprise or consists of a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,

98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81.

That is, the scFv light and heavy chains may consist entirely of the peptide sequence defined above or the scFv light and heavy chains may comprise the peptide sequence defined above, wherein the scFv light and heavy chains may contain additional peptide sequences.

In certain embodiments, the scFv light chain of the first linker comprises or consists of the VL antigen-binding domain of the 4D5 antibody and the scFv heavy chain of the first linker comprises or consists of the VH antigen-binding domain of the 4D5 antibody.

In certain embodiments, the scFv light chain of the third linker comprises or consists of the VL antigen-binding domain of the 4D5 antibody and the scFv heavy chain of the third linker comprises or consists of the VH antigen-binding domain of the 4D5 antibody.

[00140] In the context of this specification, the term 4D5 refers to the humanized monoclonal antibody trastuzumab, also known as Herceptin, and also referred to herein as "TZB" which is directed against the proximal domain of the membrane IV of HER2 (Cho et al ., 2003).

In certain embodiments, the scFv linker chain comprises a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID NO.16, wherein more particularly the scFv linker chain comprises a peptide sequence identical to SEQ ID NO.16.

[00142] In certain embodiments, the scFv linker chain with-

comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, T5%, 16%, TT%, 78%, 79%, 80%, 81%, 82 %, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No.16.

In certain embodiments, the first polypeptide chain comprises a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.50 and SEQ ID NO. 76, wherein more particularly the first polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO 41, SEQ ID NO 50 and SEQ ID NO. 76, and the second polypeptide chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a sequence of peptide selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.51, SEQ ID NO.52 and SEQ ID 77, wherein more particularly the second polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO. 29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.51, SEQ ID NO.52, and SEOQ ID. 77.

In certain embodiments, the first polypeptide chain comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO 41, SEQ ID NO 50 and SEQ ID NO. 76, and the second polypeptide chain comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40 , SEQ ID NO 42, SEQ ID NO 51, SEQ ID NO 52 and SEQ ID NO. 77.

In certain embodiments, the third polypeptide chain comprises a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO.50 and SEQ ID NO. 76, wherein more particularly the third polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO 41, SEQ ID NO 50 and SEQ ID NO. 76, and the fourth polypeptide chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a sequence of peptide selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.51, SEQ ID NO.52 and SEQ ID 77, wherein more particularly the fourth polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.51, SEQ ID NO.52 and SEOQ ID. 77.

In certain embodiments, the third polypeptide chain comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO 41, SEQ ID NO 50 and SEQ ID NO. 76, and the fourth polypeptide chain comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40 , SEQ ID NO 42, SEQ ID NO 51, SEQ ID NO 52 and SEQ ID NO. 77.

In certain embodiments, the first polypeptide chain and the third polypeptide chain each comprise a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO.41 , SEQ ID No. 50 and SEQ ID. 76, wherein more particularly the first polypeptide chain and the third polypeptide chain each comprise a peptide sequence identical to a peptide sequence selected from SEQ ID No. 18, SEQ ID No. 30, SEQ ID No. 31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQID No. 50 and SEQ ID No. 76 and the second polypeptide chain and the fourth polypeptide chain each comprise a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95 % or more, with a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID. 77, wherein more particularly the second polypeptide chain and the fourth polypeptide chain each comprise a peptide sequence identical to a peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27 , SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.51, SEQ ID NO.52, and SEQ ID NO. 77.

[00148] In certain embodiments, the first polypeptide chain and the third polypeptide each comprise a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, T5%, 76%, TT%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 50 and SEQ ID No. 76, and the second polypeptide chain and the fourth polypeptide chain.

tip each comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO. 42, SEQ ID NO.51, SEQ ID NO.52 and SEOQ ID. 77.

Therefore, the VL and VH antigen-binding domain of the first, second, third and fourth polypeptide chains may be substantially identical to the VL and VH antigen-binding domains of the scFv fragment termed A21 (Hu S. et a/. , 2008, Proteins, 70, 938-949) that specifically binds to the domain | of HER2.

In certain embodiments, the first polypeptide chain comprises a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.78, wherein more particularly the first polypeptide chain comprises a peptide sequence identical to SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.388 and SEQ ID No. 78, and the second polypeptide chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.79, wherein more particularly the second polypeptide chain comprises a peptide sequence identical to SEQ ID NO.33, SEQ ID NO.34, SEQ ID No. 35 and SEQ ID No. 79.

In certain embodiments, the first polypeptide chain comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQID No. 78, and the second polypeptide chain comprises a peptide sequence having a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86% , 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79.

In certain embodiments, the third polypeptide chain comprises a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.38, and SEQ ID NO.78, wherein more particularly the third polypeptide chain comprises a peptide sequence identical to SEQ ID NO.36, SEQ ID NO.37, SEQ ID NO.388 and SEQ ID No. 78, and the fourth polypeptide chain comprises a peptide sequence having a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.79, wherein more particularly the fourth polypeptide chain comprises a peptide sequence identical to SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID No. 79.

In certain embodiments, the third polypeptide chain comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%,

85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQID No. 78, and the fourth polypeptide chain comprises a peptide sequence having a sequence identity of 70%, 71%, 72%, 73%, T4%, 75% , T6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92 %, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.79.

In certain embodiments, the first polypeptide chain and the fourth polypeptide chain each comprise a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more , even more particularly 95% or more, with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, wherein more particularly the first polypeptide chain and the fourth polypeptide chain each comprise a sequence - peptide sequence identical to SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQID No. 78, and the second polypeptide chain and the fourth polypeptide chain each comprise a peptide sequence with an identity of sequence of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No. 79, in that more particularly the second polypeptide chain and the fourth polypeptide chain each buy end a peptide sequence identical to SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.79.

[00155] In certain embodiments, the first polypeptide chain and the third polypeptide chain.

tip each comprises a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, and the second polypeptide chain and the fourth polypeptide chain each comprise a peptide sequence with a sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84% , 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 33, SEQ ID No. 34, SEQ ID No. 35 and SEQ ID No.

79.

Therefore, the immunoglobulin light and heavy chains of the first, second, third and fourth polypeptide chains may comprise IgG domains (VL, CL, VH, CH1, CH2 and/or CH3) substantially identical to the corresponding domains of the antibody ErbB2 termed 7C2 (US 7,371,376), which specifically binds to the domain | of HER2.

In certain particular embodiments, the tetrameric polypeptide according to the invention is essentially an antibody of the immunoglobulin G type (particularly a human or humanized IgG monoclonal antibody) having two identical heavy chains and two identical light chains, in that the antigen-specific variable heavy and light chains together form a linker (the second and fourth linkers) specifically reactive to the D1 domain of Her2, and that each of the light chains, or each of the heavy chains, contains a linked polypeptide at the N-terminus comprising an scFv polypeptide chain made up of a heavy and light variable region linked by an scFv linker chain, and the scFv polypeptide chain is linked to the N-terminus of the heavy or light chain of immunoglobulin by means of an interdomain amino acid linker consisting of 1 to 20 amino acids.

In certain embodiments, the first polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 1, in which more particularly the first polypeptide chain is identical to SEQ ID No. 1.

the second polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 2, in which more particularly the second polypeptide chain is identical to SEQ ID No. 2.

[00159] In certain embodiments, the first polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 1 and the second polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.

two.

[00160] In certain embodiments, the third polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 1, in which more particularly the third polypeptide chain.

tip is identical to SEQ ID No. 1.

the fourth polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 2, in which more particularly the fourth polypeptide chain is identical to SEQ ID No. 2.

In certain embodiments, the third polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 1 and the fourth polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.

two.

In certain embodiments, the first polypeptide chain and the third polypeptide chain each have a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 1, wherein more particularly the first polypeptide chain and the third polypeptide chain are identical to SEQID No. 1 and the second polypeptide chain and the fourth polypeptide chain each have a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 2, more particularly the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID No. 2.

In certain embodiments, the first polypeptide chain and the third polypeptide chain each have a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, T6%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or 100% with SEQID No. 1 and the second polypeptide chain and the fourth polypeptide chain each have a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 17%, 18%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 2.

[00164] The resulting tetrameric polypeptide is referred to as "441" (for identity). In this construct, IgG light chains (from the first and third polypeptide chains) comprising the VL antigen-binding domain of the A21 antibody are fused at the N-terminus to a scFv fragment comprising the antigen-binding domains 4D5 VL and VH (trastuzumab or HERCEPTIN, HER2 D4 connector) and combined with IgG heavy chains (the second and fourth polypeptide chains), comprising the VH antigen-binding domain of antibody A21. The VL and VH antigen-binding domains of A21 together constitute a D1 connector of HER?2.

In certain embodiments, the first polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 3, in which more particularly the first polypeptide chain is identical to SEQ ID No. 3.

the second polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with

SEQ ID No. 4, more particularly the second polypeptide chain being identical to SEQ ID No. 4.

[00166] In certain embodiments, the first polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% with SEQ ID No. 3 and the second polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.

4.

In certain embodiments, the third polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 3, in which more particularly the third polypeptide chain is identical to SEQID No. 3 and the fourth polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 4, wherein more particularly the fourth polypeptide chain is identical to SEQ ID No. 4.

[00168] In certain modalities, the third polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80% , 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97 %, 98%, 99% or 100% with SEQ ID No. 3 and the fourth polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77% , 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94 %, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.

4.

In certain embodiments, the first polypeptide chain and the third polypeptide chain each have a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 3, wherein more particularly the first polypeptide chain and the third polypeptide chain are identical to SEQID No. 3 and the second polypeptide chain and the fourth polypeptide chain each have a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 4, more particularly the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID No. 4.

[00170] In certain embodiments, the second polypeptide chain and the third polypeptide chain each have a sequence identity of 70%, 71%, 72%, 73%, T4%, T5%, 76%, 77 %, 18%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 3 and the second polypeptide chain and the fourth polypeptide chain each have a sequence identity of 70%, 71%, 72%, 73%, T4%, 75%, T6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID #4.

[00171] The resulting tetrameric polypeptide is referred to as

"47C2" (for identity). In this construct, IgG light chains (from the first and third polypeptide chains) comprising the VL antigen-binding domain of the 7C2 antibody are fused at the N-terminus to a scFv fragment comprising the VL and VH antigen-binding domains of 4D5 (trastuzumab or HERCEPTIN, HER2 D4 connector) and combined with IgG heavy chains (the second and fourth polypeptide chains) comprising the VH antigen binding domain of the 7C2 antibody. The VL and VH antigen-binding domains of 7C2 together constitute a D1 connector of HER2).

In certain embodiments, the first polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 11, in which more particularly the first polypeptide chain is identical to SEQID No. 11 and the second polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 12, wherein more particularly the second polypeptide chain is identical to SEQ ID No. 12.

In certain embodiments, the first polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, T7T%, 718%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 11 and the second polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, T7T %, 718%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.

12.

In certain embodiments, the third polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 11, in which more particularly the third polypeptide chain is identical to SEQID No. 11 and the fourth polypeptide chain has a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 12, wherein more particularly the fourth polypeptide chain is identical to SEQ ID No. 12.

In certain embodiments, the third polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77%, 78%, 79%, 80 %, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 11 and the fourth polypeptide chain has a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, 16%, 77 %, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 291%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID NO.

12.

In certain embodiments, the first polypeptide chain and the third polypeptide chain each have a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 11, wherein more particularly the first polypeptide chain and the third polypeptide chain are identical to SEQID No. 11 and the second polypeptide chain and the fourth polypeptide chain each have a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 12, where more particularly the second polypeptide chain and the fourth polypeptide chain are identical to SEQ ID No. 12.

In certain embodiments, the second polypeptide chain and the third polypeptide chain each have a sequence identity of 70%, 71%, 72%, 713%, T4%, T5%, 16%, 77 %, 18%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID No. 11 and the second polypeptide chain and the fourth polypeptide chain each have a sequence identity of 70%, 71%, 72%, 73%, T4%, 15%, T6%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87% , 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% with SEQ ID #12.

The resulting tetrameric polypeptide is referred to as "241" (for identity). In this construct, IgG heavy chains (from the second and fourth polypeptide chains) comprising the VH antigen-binding domain of the A21 antibody are fused at the N-terminus to a scFv fragment comprising the VL and antigen-binding domains 4D5 VH (trastuzumab or HERCEPTIN, HER2 D4 connector) and combined with IgG light chains (the first and third polypeptide chains) comprising the VL antigen binding domain of antibody A21. The VL and VH antigen-binding domains of A21 together constitute a D1 connector of HER?2.

[00179] In certain embodiments, the VH antigen binding domain is selected from the VH antigen binding domain of A21 (particularly SEQ ID NOs. 40, 42, 51, 52 or 77) and the VH antigen binding domain of 7C2 (particularly SEQ ID No. 79) and wherein the VL antigen binding domain is selected from the VL antigen binding domain of A21 (particularly SEQ ID No. 39, 41, 50 or 76) and the binding domain to the 7C2 VL antigen (particularly SEQ ID No. 78).

[00180] In certain embodiments, the first and third polypeptide chains are identical to SEQ ID No. 1 or an equivalent functional peptide sequence with a sequence identity of at least 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98% or 99%, and the second and fourth polypeptide chains are identical to SEQ ID No. 2 or an equivalent functional peptide sequence with a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% (construct 441 in the case of sequence identity).

[00181] In certain embodiments, the first and third polypeptide chains are identical to SEQ ID No. 3 or an equivalent functional peptide sequence with a sequence identity of at least 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98% or 99%, and the second and fourth polypeptide chains are identical to SEQ ID No. 4 or an equivalent functional peptide sequence with a sequence identity of at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% (construct 47C2 in the case of sequence identity).

[00182] In certain embodiments, the first and third polypeptide chains are identical to SEQ ID No. 11 or an equivalent functional peptide sequence with a sequence identity of at least 70%, 75%, 80%, 85 %, 90%, 95%, 96%, 97%, 98% or 99%, and the second and fourth polypeptide chains are identical to SEQ ID No. 12 or an equivalent functional peptide sequence with a sequence identity of at least 70%, 75%, 80%, 85%, 90%,

95%, 96%, 97%, 98% or 99% (construct 241 in the case of sequence identity).

The interdomain amino acid linker is not restricted in amino acid composition, but amino acids that contribute to the flexibility of the linker are chosen in the particular embodiments contemplated herein. The inventors showed that the linkers work consisting of residues G, S and/or T, for example, repeats of (GGmS) and (GGmT) with m selected from 1 to 3, and the entire length of the linker not exceeding 20, 25 or even 30. Interdomain linkers with only one or two amino acids have been shown to work. The first and third polypeptides can comprise the same interdomain amino acid linker or different interdomain amino acid linkers.

In certain embodiments, the interdomain amino acid linker consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24 or 25 amino acids.

[00185] — With regard to the length and sequence composition of the interdomain amino acid linker, the inventors' results indicate that any linker with an equivalent length of a maximum of 25 amino acids would not be expected to, because the structure prediction interferes with the solubility of the resulting protein, it is expected to work.

[00186] The polypeptide, according to any of the above embodiments, wherein the interdomain amino acid linker comprises or consists of the amino acids G, A, J, S, TT, P, C, V, MeFE, particularly in that the interdomain amino acid linker comprises or consists of amino acids G, S, A, and T.

[00187] In particular embodiments, the interdomain amino acid linker is (GG))) with n being an integer and n 2 4 (particularly n is 4, 5, 6, 7 or 8), and with selected from S and T.

[00188] In particular embodiments, the interdomain amino acid linker is (GGS), with n being an integer and n being 2 4 (particularly n being 4, 5, 6,7 or 8).

[00189] In particular embodiments, the interdomain amino acid linker is (GGT), with n being an integer and n being 2 4 (particularly n being 4, 5, 6,7 or 8).

[00190] In particular embodiments, the interdomain amino acid linker is (GXG), with n being an integer and n 2 4 (particularly n is 4, 5, 6, 7 or 8), and with Z selected from S and T.

[00191] In particular embodiments, the interdomain amino acid linker is (TTZ), with n being an integer and n 2 4 (particularly n being 4, 5, 6, 7, or 8), and with each Iº independently of any other Iº being selected from A, Ge V, and Z being selected from Se T.

[00192] “Important considerations when choosing the binder sequence were solubility and flexibility. One skilled in the art will readily be able to vary this sequence in composition and length, based on the teaching contained herein and available knowledge about binder design, as exemplified by Chen et al., 2013, Advanced Drug Delivery Reviews, 65, 1357-1369 and Evers et al., 2006, Biochemistry, 45, 13183-13192.

[00193] In certain embodiments, the interdomain amino acid linker is characterized by an amino acid sequence (GGGGS), with n being 1,2,3,4or5.

In certain embodiments, the inter-domain amino acid linker comprises or is a sequence having features of one of SEQ ID NO.17, SEQ ID NO.55 to SEQ ID NO.69, and SEQ ID NO.82 to SEQ ID NO. 91.

[00195] In certain embodiments, the inter-domain amino acid linker comprises or consists of a sequence of se-

selected from one of SEQ ID NO.17, SEQ ID NO.55 to SEQ ID NO.69, and SEQ ID NO.82 to SEQ ID NO.91 or an equivalent functional peptide sequence with a sequence identity of at least 70%.

A second aspect of the invention relates to the polypeptide according to the first aspect for use in a method for the prevention or treatment of a malignant neoplastic disease associated with the expression of HER2 (a cancer positive for HER2).

[00197] Likewise, a dosage form for the prevention or treatment of a malignant neoplastic disease associated with the expression of HER2 comprising a tetrameric polypeptide of the invention is provided.

[00198] The skilled person is aware that any drug specifically mentioned may be present as a pharmaceutically acceptable salt of said drug. Pharmaceutically acceptable salts comprise the ionized drug and an oppositely charged counterion. Non-limiting examples of pharmaceutically acceptable anionic salt forms include acetate, benzoate, besylate, bitatrate, bromide, carbonate, chloride, citrate, edetate, edisylate, embonate, estolate, fumarate, gluceptate, gluconate, hydrobromide, hydrochloride, iodide , lactate, lactobionate, malate, maleate, mandelate, mesylate, methyl bromide, methyl sulfate, mucote, napsylate, nitrate, pamoate, phosphate, diphosphate, salicylate, disalicylate, stearate, succinate, sulfate, tartrate, tosylate - to, triethodide and valerate. Non-limiting examples of pharmaceutically acceptable cationic salt forms include aluminum, benzathine, calcium, ethylenediamine, lysine, magnesium, meglumine, potassium, procaine, sodium, tromethamine and zinc.

The dosage forms may be for enteral administration, such as nasal, buccal, rectal, transdermal or oral administration, or as an inhalation or suppository form. Alternatively, parenteral administration can be used, such as subcutaneous, intravenous, intrahepatic or intramuscular injection forms. Optionally, a pharmaceutically acceptable carrier and/or excipient can be present.

[00200] Topical administration is also within the scope of the advantageous uses of the invention. The person skilled in the art is aware of a wide range of possible recipes for delivering topical formulations, as exemplified by the contents of Benson and Watkinson (Eds.), Topical and Transdermal Drug Delivery: Principles and Practice (1st Edition, Wiley 2011, ISBN-13) : 978-0470450291); and Guy and Handcraft: Transdermal Drug Delivery Systems: Revised and Expanded (2nd Ed., CRC Press 2002, ISBN-13: 978-0824708610); Osborne and Amann (Eds.): Topical Drug Delivery Formulations (1st Ed. CRC Press 1989; ISBN-13: 978-0824781835).

A third aspect of the invention relates to an isolated nucleic acid encoding at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the tetrameric polypeptide, according to with the first aspect of the invention. In particular, the isolated nucleic acid can be comprised on a plasmid for expression in a bacterial or eukaryotic host cell. Nucleic acid sequences encoding the first, second, third, and fourth polypeptide can be provided on the same plasmid or on separate plasmids, i.e., and for co-expression in the same host.

A fourth aspect of the invention relates to a host cell that is adapted to produce at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the tetrameric polypeptide, according to with the first aspect of the invention. In part

cellular, the host cell is a bacterial cell or a eukaryotic cell. More particularly, the host cell is a Chinese hamster ovary (CHO) cell.

In certain embodiments, the host cell comprises isolated nucleic acid according to the third aspect of the invention, such that the host cell is capable of producing at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide, according to the first aspect of the invention. In particular, the first, second, third and fourth polypeptides can be co-produced in the same cell or produced separately and combined in vitro.

[00204] "A fifth aspect of the invention relates to a method for obtaining the polypeptide according to the first aspect of the invention, wherein the method comprises culturing the host cell according to the fourth aspect of the invention, of so that at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide, according to the first aspect of the invention, is produced. Pharmaceutical composition and administration

[00205] "Another aspect of the invention relates to a pharmaceutical composition comprising a tetrameric polypeptide of the present invention, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier. In additional embodiments, the composition comprises at least two pharmaceutically acceptable carriers as described herein.

In certain embodiments of the invention, the tetrameric polypeptide of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controlled dosage.

drug and to give the patient an elegant and easy-to-handle product.

[00207] In embodiments of the invention related to topical uses of the tetrameric polypeptide of the invention, the pharmaceutical composition is formulated in a manner that is suitable for topical administration, such as aqueous solutions, suspensions, ointments, creams, gels or sprayable formulations, by for example, for aerosol delivery or the like, comprising the active ingredient together with one or more of the solubilizers, stabilizers, tonicity enhancing agents, buffers and preservatives that are known to those of skill in the art.

The pharmaceutical composition can be formulated for oral administration, parenteral administration or rectal administration. Furthermore, the pharmaceutical compositions of the present invention can be made in solid form (including, without limitation, capsules, tablets, pills, granules, powders or suppositories), or in liquid form (including, without limitation, solutions, suspensions or emulsions).

[00209] The dosage regimen for the compounds of the present invention varies depending on known factors such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the species, age, sex, health, medical condition, and weight of the recipient; the nature and extent of symptoms; the type of concomitant treatment; the frequency of treatment; the route of administration, the renal and hepatic function of the patient and the desired effect. In certain embodiments, the compounds of the invention can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times a day.

[00210] The pharmaceutical compositions of the present invention may be subjected to conventional pharmaceutical operations, such as sterilization and/or may contain conventional inert diluents,

lubricating agents or buffering agents, as well as adjuvants such as preservatives, stabilizers, wetting agents, emulsifiers and buffers etc. They can be produced by conventional processes, for example by conventional mixing, granulating, dissolving or lyophilizing processes. Many such procedures and methods for preparing pharmaceutical compositions are known in the art, see, for example, L. Lachman et al. The Theory and Practice of Industrial Pharmacy, 4th Ed, 2013 (ISBN 8123922892).

[00211] The invention further refers to the following items, which may also be formulated as claims:

[00212] item 1: A tetrameric polypeptide comprising or consisting of

[00213] - a first polypeptide chain comprising a first linker that binds to a D4 epitope of HER2, an interdomain amino acid linker and a first immunoglobulin domain,

[00214] - a second polypeptide chain comprising a second immunoglobulin domain, wherein the first immunoglobulin domain and the second immunoglobulin domain, together, constitute a second linker, particularly a Fab domain, which binds to a D1 epitope of HER?2,

[00215] - a third polypeptide chain comprising a third linker that binds to a D4 epitope of HER2, an interdomain amino acid linker and a third immunoglobulin domain, and

- a fourth polypeptide chain comprising a fourth immunoglobulin domain, wherein the third immunoglobulin domain and the fourth immunoglobulin domain together constitute a fourth linker, particularly a Fab domain, which binds to a D1 epitope of HER?2.

Item 2: The polypeptide according to item 1, wherein said first immunoglobulin domain is substantially the same as said third immunoglobulin domain and/or wherein said second immunoglobulin domain is substantially the same same as said fourth immunoglobulin domain.

[00218] ltem3: The polypeptide according to item 1 or 2, wherein said first linker is substantially the same as said third linker.

[00219] Item 4: The polypeptide according to any of the foregoing items, wherein said first linker and/or said third linker comprises or consists of a single chain variable fragment polypeptide chain comprising one scFv heavy chain, one scFv linker chain and one scFv light chain.

Item 5: The polypeptide according to item 4, wherein said scFv heavy chain is the VH domain of 4D5 and wherein said scFv light chain is the VL domain of 4D5.

[00221] Item 6: The polypeptide according to any one of items 1 to 5, wherein

- said first immunoglobulin domain is a VL domain and said second immunoglobulin domain is a VH domain and/or wherein said third immunoglobulin domain is a VLe domain said fourth immunoglobulin domain is a VH domain; or

[00223] - said first immunoglobulin domain is a VH domain and said second immunoglobulin domain is a VL domain and/or wherein said third immunoglobulin domain is a VHe domain said fourth immunoglobulin domain is a VL domain.

[00224] item 7: The polypeptide according to item 6, wherein said VH domain is linked, at the C-terminus, to a CH1 domain.

[00225] Item 8: The polypeptide according to item 7, wherein said CH1 domain is linked, at the C-terminus, to a CH2 domain or a CH2 domain and a CH3 domain.

[00226] Item 9: The polypeptide according to any one of items 6 to 8, wherein said VL domain is linked, at the C-terminus, to a CL domain.

[00227] Item 10: The polypeptide according to any one of items 6 to 9, wherein said VH domain is selected from the VH domain of A21 and the VH domain of 7C2, and wherein said VL domain is selected from the VL domain of A21 and the VL domain of 7C2.

[00228] Item 11: The polypeptide according to any of the above items, wherein said interdomain amino acid linker consists of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids.

[00229] Item 12: The polypeptide according to any of the preceding items, wherein said interdomain amino acid linker comprises or consists of amino acids G, A, J, S, TT, P, C, V, MeFE, particularly wherein said interdomain amino acid linker comprises or consists of amino acids G, S, A and T.

[00230] Item 13: The polypeptide according to any of the preceding items, wherein said interdomain amino acid linker is characterized by an amino acid sequence (GGGGS)r, with n being 1,2,3,4 or 5.

Item 14: The bispecific HER2 polypeptide according to any of the above items, wherein said interdomain amino acid linker comprises or is a sequence having characteristics of one of SEQ ID. 17, SEQ ID. 55 the SEQ ID. 69 and SEQ ID. 82 the SEQ ID. 91.

[00232] Item 15: The polypeptide according to any of the preceding items, wherein said interdomain amino acid linker comprises or consists of a peptide sequence selected from one of the SEQ ID. 17, SEQ ID. 55 the SEQ ID. 69 and SEQ ID. 82 the SEQ ID. 91 or an equivalent functional peptide sequence with a sequence identity of at least 70%.

[00233] Item 16: The polypeptide, according to any of the above items, for use in a method for the prevention or treatment of a malignant neoplastic disease associated with the expression of Her2.

Wherever alternatives to individual separable characteristics, such as, for example, a protein isotype or coding sequence, type of linker or medical indication are presented here as "modalities", it should be understood that such alternatives may be freely combined to form discrete embodiments of the invention described herein. Thus, any of the alternative modalities for a detectable marker can be combined with any of the alternative ligand modalities, and these combinations can be combined with any medical indication or diagnostic method mentioned herein.

[00235] The invention is further illustrated by the following examples and figures, from which other embodiments and advantages can be drawn. These examples are intended to illustrate the invention, but not to limit its scope. Description of Figures

[00236] Fig 1 shows schematics of biparatopic anti-HER?2 binding agents.

[00237] AfFig 2 shows other schematics of biparatopic anti-HER2 binding agents

[00238] Fig.3 shows a vector map of a plasmid for co-expression of the light and heavy chain of construct 441 in CHO cells (Pymex10-based vector with double expression cassette [CMV GO! polyA]).

[00239] Fig.4 shows a vector map of a plasmid for co-expression of the light and heavy chain of the 47C2 construct in CHO cells (Pymex10-based vector with dual expression cassette [CMV GO! polyA]).

[00240] Fig.5 shows a vector map of a plasmid for the expression of construct 841 in CHO cells.

[00241] Fig. 6 shows an elution profile of construct 441 from Protein A affinity chromatography.

[00242] Fig 7 shows an elution profile of construct 441 from cation exchange chromatography.

[00243] Fig 8 shows an elution profile of construct 441 from size exclusion chromatography.

[00244] Fig.9 shows a Coumassie-stained SDS-PAGE gel of fractions from the purification of construct 441.

[00245] Fig.10 shows the CHO viability test during the expression of the 441 construct (scFV-IgG). Optimization of construct 441 expression in CHO cells for the indicated time. Cells were cultivated in Mlrus' CHOgro medium (MIR 6260) and additionally fed with free cysteine (reduced form) (2), glutathione (3), fetal calf serum (4) or all additives, respectively ( 5). CHO cells were analyzed in the CASY cell counter (Schärfe System).

[00246] Fig 11 shows a Western blot of the expression of construct 441, secreted into the medium of CHO cells after the indicated times. Cells were grown in Mlirus' CHOgro medium (MIR 6260) (1) and additionally fed with free cysteine (reduced form) (2), glutathione (3), fetal calf serum (4) or all additives together ( 5), respectively. Protein was precipitated from the medium by precipitation with acetone and resolubilized in SDS PAGE buffer. Proteins were resolved on a 4-12% gradient gel and the Western blot was analyzed on an Odyssey system (LI-COR). The purified intact full-length construct 441 is shown as a control (A) and runs above the 170 kDa marker. Thermo Scientific Page ruler molecular weight marker is shown in red.

[00247] Fig. 12 shows cell proliferation assays (XTT)

with BT474 cells after 4 days of treatment. Trastuzumab (TZB), biparatopic DARPin (6L1G) and different fusion variants of the biparatopic construct. LF IgG HL (murine parent of construct 441), HF IgG HL (murine parent of construct 241) show similar antiproliferative activity compared to biparatopic DARPin 6L1G, which is superior to trastuzumab (TZB). HF IgG LH (murine variant, no seq.) and LF IgG LH (murine variant, no seq.) show reduced antiproliferative activity compared to biparatopic DARPin and higher ICso concentrations.

[00248] Fig.13 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment to test the effect of ligand length. Biparatopic DARPin (6L1G) and different fusion ligand variants of the biparatopic construct (murine parent construct of 441) are compared. The 2-AA ligand (GS) shows the greatest antiproliferative activity. Ligands 4-, 7- and 12-AA show similar activity. The 22-AA ligand variant shows reduced activity.

[00249] Fig. 14 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441), biparatopic construct 411 (humanized VH1 kappa1l) and biparatopic construct 443 (humanized VH3 kappa4). All show similar plateau levels of antiproliferative activity, except 443, which shows reduced activity.

[00250] Fig.15 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment with different humanized versions of IgG A21, when fused to TZB scFv. The humanization strategy is described above. Different variants use humanized kappa1 VH3 or a humanized kappa1 VH core graft.

[00251] Fig.16 shows the XTT cell proliferation assay with BT474 cells after 4 days of treatment. Tetravalent IgG (murine HF IgG HL and murine LF IgG HL) versus bivalent Fab fusions (murine HF Fab HL and murine LF Fab HL). All constructs show similar plateau and IC50 values.

[00252] Fig. 17 shows the XTT cell proliferation assay with SKBR3 cells after 4 days of treatment. Biparatopic DARPin (6G) biparatopic construct (441 tf), trastuzumab (TZB).

[00253] Fig.18 shows cell proliferation assays (XTT) with CALU-3 cells after 4 days of treatment. Biparatopic DARPin (6G), biparatopic construct (construct 441 (441tf), trastuzumab (TZB).

Fig.19 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment testing the effect of domain 1 binding unit. Biparatopic construct with A21 (construct 441tf) or 7C2 fusions show different IC50 and plateau level.

[00255] Fig.20 shows cell proliferation assays (XTT) with BT474 cells after 4 days of treatment testing the effect of domain 1 binding unit. Biparatopic construct with A21 (construct 441) or with 398 ( 39s HF IgG HL)

[00256] Fig.21 shows XTT cell proliferation assays with HCC1419 cells after 4 days of treatment. Biparatopic DARPin (6G; 6L1G), biparatopic construct 441 (441tf) and bivalent LF-oaFabFc (A21-TZB-40a). 441 and 6G show similar inhibition of cell proliferation after 4 days. LF-oaFabFc shows slightly reduced inhibition of cell proliferation compared to 441.

[00257] Fig. 22 shows the XTT cell proliferation assay with BT474 and HCC1419 cells after 4 days of treatment. All human.

[00258] A-Fig. 23 shows the XTT cell proliferation assay with BT474 and HCC1419 cells after 4 days of treatment. All human.

[00259] Fig.24 shows a) in the upper panel, the XTT cell proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 days of treatment; and in the lower panel, XTT cell proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 days of treatment (higher affinity variants (NGS and GGG)); b) repeated experiments with a new NGS expression.

[00260] Fig.25 shows XTT cell proliferation assays with BT474 (left) and HCC1419 (right) cells after 4 days of treatment.

[00261] Fig.26 shows XTT cell proliferation assays with HCC1419 cells cultured as 3D spheroids.

[00262] Fig.27 shows Western Blots 24 hours after treatment (BT474) with the indicated agents (murine).

[00263] Fig.28 shows in the upper panel the induction of apoptosis in BT474 cells after 3 days of treatment. The mean number of positive cells for propidium iodide (PI) was determined for 4 repetitions, counted by a cell profiler and analyzed with Student's t test. The biparatopic construct (441, 441tf) induced significantly more cell death than trastuzumab (TZB). 441 and biparatopic DARPin (6L1G) show a similar level of cell death; and in the lower panel, the induction of apoptosis in BT474 cells after 3 days of treatment. The mean number of annexin-V positive cells was determined for 3-4 replicates, counted by the cell profiler and analyzed with Student's t-test. The biparatopic construct 441 induced significantly more apoptosis than trastuzumab (TZB). Construct 441 and 6L1G show a similar level of apoptosis.

Fig 29 shows images of BT474 cells treated with the indicated agents for 3 days.

[00265] Fig 30 shows Alexa647-labeled trastuizumab (TZB), biparatopic construct 441 and biparatopic constructs of oaLF and oaHF arm constructs were incubated for 1 h concentration of 100 nM with 3 million BT474 cells in 3 ml of PBS containing NaN;3 (0.1%) and BSA (1%) at 4°C. Note that BT474 cells were pretreated with 0.1% NaN;3 in PBS with 1% BSA to block internalization prior to binding. The cells were later analyzed using the CyFlow Space instrument (Partec). All binding agents show specific binding to the surface of HER?2 positive BT474 cells.

[00266] Fig.31 shows the induction of cell death after treatment with 100 nM of the indicated agents. BT474, N87, HCC1419 and SKBR3 cells were seeded 24 h before treatment in 96 black clear well microscopy plates (Nunc), continuously treated for 3 days and stained with HOECHST-33342 (Invitrogen) for whole cells and with propidium iodide (Sigma) for dead cells permeable to the membrane. The cells were analyzed in a Lionheart FX automated microscope (BioTek Instruments) and the number of positive cells for propidium iodide and HOECHST-33342 was quantified with Gen5 software (BioTek Instruments). The ratio of positive cells for propidium iodide and HOECHST-33342 was calculated for 3 biological replicates and the mean and SD are shown in the corresponding column graphs. Biparatopic binding agents (6L1G, 441, 841, LFoa, 241, 641, HFoa, 7C2LF) that bind to domain 1 and 4 of HER2 continuously induce more dead cells than trastuzumab (TZB) or the trastuzumab combination and pertuzumab (TZB + PZB) in HER2-positive cancer cells.

[00267] Fig.32 shows the half-life of the 441 construct in the serum of NSG mice. Sera collected from mice with 441 in-

Previous injections (3 mg/kg) were analyzed by sandwich ELISA. 441 showed an alpha phase of about 4.3 hours, followed by a beta phase of more than 45 hours.

[00268] Fig.33 shows the in vivo activity of 441 in N87 xenografts in beige SCID mice. After N87 tumors reached 150 mm? of size, mice were treated with eight injections of 441 (10 mg/kg) for four weeks. 441 led to a significant reduction in tumor size compared to untreated mice and mice treated with TZB (10 mg/kg) or huA21G (10 mg/kg).

[00269] Fig.34 shows representative microscopy images of BT-474 cells after treatment for 2 hours with trastuzumab (TZB), huA21G (A21), their combination or 441, and untreated cells, without or with addition of an antibody IM anti-human such as controls. Nuclei were stained using 2-(4-amidinophenyl)-1H-indole-6-carboximidamide (DAPI), antibodies were detected with a goat anti-human Fc antibody and lysosomal compartments using an anti-LAMP1 antibody.

[00270] Fig.35 shows the result of a treatment over time and subsequent surface protein internalization and degradation assay for constructs 441 and 841, hA21G, trastuzumab (TZB), the combination of trastuzumab and hA21G (TZB + hA21G), pertuzumab (PZB), the combination of trastuzumab and pertuzumab (TZB + PZB), and the HSP90 inhibitor geldanamycin (GA). Sequence listing: SEQ ID No. 1 ("441 1 polypeptide"):

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGCGGESCGGGESGÇE GGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQOPEDFATYYCQQ HYTTPPTFGQGTKVEIKGSDIVLTOSPDSLAVSLGERATINCRSSQTL LYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKSGVPDRFSGSGSGT DFTLTISSLOAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVEFI FPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN

RGEC SEQ ID No. 2 ("441 2 polypeptide"):

QVQALVASGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL ESVVTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 3 ("47C2 1 polypeptide"):

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSCGGGSCGGGSE GGGSDIQMTASPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQOPEDFATYYCQQ HYTTPPTFGQGTKVEIKGSDIVMTQOSPDSLAVSLGERATINCRASQSV SGSRFTYMHWYQQKPGQPPKLLIKYASILESGVPDRFSGSGSGTDFT LTISSLOAEDVAVYYCQHSWEIPPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG EC

SEQ ID No. 4 ("47C2 2 polypeptide"):

EVQLVOSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQOG LEWIGMIHPLDAEIRANQKFRDRVTITVDTSTSTAYLELSSLRSEDTAV YYCARGTYDGGFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLESV VTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 5 ("841 2 Polypeptide"):

QVQALVASGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL

ESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 6 ("87C2 polypeptide 2"):

EVQLVOSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQOG LEWIGMIHPLDAEIRANQKFRDRVTITVDTSTSTAYLELSSLRSEDTAV YYCARGTYDGGFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLESV

VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 7 ("841Fc 1 polypeptide"):

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSCGGGSCGGGSE GGGSDIQMTASPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQOPEDFATYYCQQ HYTTPPTFGQGTKVEIKGSDIVLTOSPDSLAVSLGERATINCRSSQTL LYSNNQKNYLAWYQKKPGQPPKLLISWAFTRKSGVPDRFSGSGSGT DFTLTISSLOAEDVAVYYCQQYSNYPWTFGQGTKVEIKRTVAAPSVEFI FPPSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESV TEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFN RGECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRK EMTKNQVLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDG

SFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 8 ("841Fc 2 polypeptide"):

QVQALVASGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL ESVVTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 9 ("87C2Fc 1 polypeptide"):

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSCGGGSCGGGSE GGGSDIQMTASPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQOPEDFATYYCQQ HYTTPPTFGQGTKVEIKGSDIVMTQOSPDSLAVSLGERATINCRASQSV SGSRFTYMHWYQQKPGQPPKLLIKYASILESGVPDRFSGSGSGTDFT LTISSLOAEDVAVYYCQHSWEIPPWTFGQGTKVEIKRTVAAPSVFIFP PSDEQLKSGTASVKCLLNNFYPREAKVQWKVDNALQSGNSQESVTE QDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRG ECDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHOQ DWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRKEM TKNQVLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLKSDGSFF

LYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 10 ("87C2Fc 2 polypeptide"):

EVQLVOSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQOG LEWIGMIHPLDAEIRANQKFRDRVTITVDTSTSTAYLELSSLRSEDTAV YYCARGTYDGGFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLESV VTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 11 ("241 1 polypeptide"):

DIVLTASPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPG QPPKLLISWAFTRKSGVPDRFSGSGSGTDFTLTISSLOAEDVAVYYC QQYSNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHOQGLSSPVTKSFNRGEC SEQ ID No. 12 ("241 2 polypeptide"):

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSCGGGSCGGGSE GGGSDIQMTASPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQOPEDFATYYCQQ HYTTPPTFGOGTKVEIKGSQVQALVASGAEVKKPGASVKVSCKASGY SFTGYFINWVREAPGQGLEWMGHISSSYATSTYNQKFQGRVTFTVD TSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLOSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

SK SEQ ID No. 13 ("641 2 polypeptide"):

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGCGGESCGGGESGÇE GGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQ HYTTPPTFGQGTKVEIKGSQVALVASGAEVKKPGASVKVSCKASGY SFTGYFINWVREAPGQGLEWMGHISSSYATSTYNQKFQGRVTFTVD TSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLOSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTK

VDKRVEPKSC SEQ ID No. 14 ("L-chain (light) of scFv TZB"):

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLL IYSASFLYSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHYTTP

PTFGQGTKVEIK SEQ ID No. 15 ("H (heavy) chain of scFv TZB"):

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSS

SEQ ID No. 16 ("ScFv TZB Linker"):

GGGEGESCGSEGSCEGESCGGGES SEQ ID No. 17 ("Linker B1 to B2 (link 1)"):

GS SEQ ID No. 18 ("L-chain of A21"):

DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPG QPPKLLISWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYSNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 19 ("H-Link of A21 with Fc"):

QVALVASGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGOQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL ESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 20 ("H-Link from A21 to Fab"):

QVALVASGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGOQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL

ESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 21 (,TZB CDR H1"):

GFNIKDTYIH SEQ ID No. 22 (,TZB CDR H2"):

RIYPTNGYTRYADSVKG SEQ ID No. 23 (,TZB CDR H3"):

WGGDGFYAMDY SEQ ID No. 24 (,TZB CDR L1"):

RASQDVNTAVA SEQ ID No. 25 (,TZB CDR L2"):

SASFLYS SEQ ID No. 26 (,TZB CDR L3"):

QQHYTTPPT SEQ ID No. 27 (.A21 CDR H1"):

GYSFTGYFIN SEQ ID No. 28 (.A21 CDR H2"):

HISSSYATSTYNQKFQG SEQ ID No. 29 ("A21 CDR H3"):

SGNYEEYAMDY SEQ ID No. 30 (.A21 CDR L1"):

RSSQTLLYSNNQKNYLA SEQ ID NO.31 (A21 CDR L2"):

WAFTRKS SEQ ID NO 32 (.A21 CDR L3"):

QQYSNYPWT SEQ ID No. 33 (.7C2 CDR H1"):

GYSFTGYWMN

SEQ ID No. 34 (.7C2 CDR H2"):

MIHPLDAEIRANQKFR SEQ ID No. 35 (.7C2 CDR H3"):

GTYDGGFEY SEQ ID No. 36 (.7C2 CDR L1"):

RASQSVSGSRFTYMH SEQ ID No. 37 (.7C2 CDR L2"):

YASILES SEQ ID NO 38 (.7C2 CDR L3"):

QHSWEIPPWT SEQ ID No. 39 ("L-chain of A21 V1º):

DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQQKPG QPPKLLISWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYSNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 40 ("H chain of A21 V1"):

QVQLVASGAEVKKPGASVKVSCKASGYSFTGYFINWVRQAPGQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 41 ("L-chain of A21 V2"):

DIVLTQSPDSLAVSLGERATINCRSSQPLEYSNNQWNYLAWYQKKPG QPPKLLISWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC GQYSDYPNTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID No. 42 ("H chain of A21 V2"):

QVALVASGAEVKKPGASVKVSCKASGYPFTQYFIHWVREAPGOQGLE WMGHISSSYATVDYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTA VYYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYS LESVVTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 43 ("L-chain of scFv TZB V1"):

DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLL IYSASFLYSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHYTTP

PTFGQGTKVEIK SEQ ID No. 44 ("H-chain of scFv TZB V1"):

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNAYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV

YYCSRWGGTGFYAMDYWGQGTLVTVSS SEQ ID No. 45 ("Alternative Fc Part - no mut"):

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 46 (,Alternative Fc Part - "Knob into hole" - site of the "knob" Vi"):

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLYCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 47 (,Alternative Fc Part - "Knob into hole" - site of "hole" Vi"):

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG FYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLTSKLTVDKSRW

QQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 48 (,Alternative Fc Part - "Knob into hole" - site of the "knob" V2"):

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVYTLPPCREEMTKNQVSLWCLVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 49 (,Alternative Fc Part - "Knob into hole" - site of "hole" V2"):

APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNW YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKV SNKALPAPIEKTISKAKGQPREPQVCTLPPSREEMTKNQVSLSCAVK GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 50 ("L-chain of A21 V3"):

DIQLTASPSSLSASVGDRVTITCRSSQTLLYSNNQKNYLAWYQQKPG KAPKLLISWAFTRKSGVPSRFSGSGSGTDFLTISSLQPEDFATYYCQ QYSNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 51 ("H chain of A21 V3"):

EVOLVOASGPELVQPGGSVRISCAASGYSFTGYFINWVKQAPGKGLE WISHISSSYATSTYNQSFKGRATFSVDTSSSTAYMQLNSLRAEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 52 ("H chain of A21 V4"):

VOLVESGGGLVQPGGSLRLSCAASGYSFTGYFINWVRQAPGKGLE WVSHISSSYATSTYNQSVKGRFTFSVDTSSSTAYLQMNSLRAEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL

SSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSC SEQ ID No. 53 ("TZB V2 H chain"):

QVALVASGAEVKKPGASVKVSCKASGFNIKDTYIHWVRQAPGQGLE QMGRIYPTNGYTRYDPKFQGRVTITADTSSNTAYMELSSLRSEDTAV

YYCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID No. 54 ("TZB V3 H-chain"):

EVOLVOASGPELVQPGGSLRLSCAASGFNIKDTYIHWVKQAPGKGLE WISRIYPTNGYTRYDPSFKGRATISADTSNTAYLQVNSLRAEDTAVY

YCSRWGGDGFYAMDYWGQGTLVTVSS SEQ ID No. 55 (Poly-Gly-Binder"):

GGGGG SEQ ID No. 56 (,Ova-linker"):

GSGSGS SEQ ID No. 57 (,Trans-linker"):

GSGGGTGGGSGÇS

SEQ ID No. 58 (,Pro-linker"):

PPP SEQ ID No. 59 (,PAS-linker 1"):

ASPAAPAPASPAAPAPSAPAA SEQ ID No. 60 (,PAS-linker 2"):

ASAAAPAAASAAAASAPSAAAA SEQ ID No. 61 (,PAS-linker 3"):

AASPAAPSAPPAAASPAAPSAPPAA SEQ ID NO 62 (,PAS-linker 4"):

ASPASA SEQ ID No. 63 (,PAS-linker 5"):

ASPASPASA SEQ ID No. 64 (.XS-linker 1"):

PAGSP SEQ ID No. 65 (,XS-linker 2"):

STEPS SEQ ID No. 66 (.XS-linker 3"):

STEEG SEQ ID No. 67 (,XS-linker 4"):

GSAPG SEQ ID No. 68 (,Proper-linker1"):

GASTP SEQ ID No. 69 (,Proper-linker2"):

GPSAT SEQ ID No. 70 (398” light chain):

DIVMTQTPLSLSVTPGQPASISCKSSQSVFFRSNNKNILAWYLQKPG QPPQLLITNWASSRESGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC QQYFGSPFTFGPGTKVDIKRTVAAPSVFIFPPSDEQLKSGTASVVCLL NNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLS

KADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 71 (,39sS heavy chain"):

EVOLVESGGGLVKPGGSLRLSCAASGFTFSSYSMSWVRQAPGKGL EWVSSISSSSSYIYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YYCARGGDAYNYYYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYS LSSVVTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 72 (.H218 light chain"):

QSALTQPASVSGSPGOSITISCTGTSSDVGGYNYVSWYQQHPGKAP KLMIYDVSKRPSGVSNRFSGSKSGNTASLTISGLQAEDEADYYCSSY TSSSTLVFGGGTKLTVLGTVAAPSVFIFPPSDEQLKSGTASVVCLLNN FYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

DYEKHKVYACEVTHOQGLSSPVTKSFNRGEC SEQ ID No. 73 ("H218 heavy chain"):

EVOLVAOSGAEVKKPGESLKISCKGSGYSFTSYWIGWVRQAPGQGLE WMGWISAYNGNTNYAQKLAGRVTMTTDTSTSTAYMELRSLRSDDTA VYYCAREGDGAFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 74 (, MF3958 light chain):

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLI YAASSLOSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCLLNNFYPRE AKVOQWKVDNALQOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 75 ("MF3958 heavy chain"):

QVALVASGAEVKKPGASVKLSCKASGYTFTAYYINWVRQAPGQGLE WIGRIYPGSGYTSYAQKFQGRATLTADESTSTAYMELSSLRSEDTAV YFCARPPVYYDSAWFAYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYS LESVVTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 76 ("L-chain of A21 V4"):

DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPG QPPKLLISWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYSNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL

SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 77 ("H chain of A21 with Fc V2"):

QVALVASGAEVKKPGASVKVSCKASGYSFTGYFINWVREAPGOQGLE WMGHISSSYATSTYNQKFQGRVTFTVDTSSSTAYMELSSLRSEDTAV YYCVRSGNYEEYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTS GGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSL ESVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFN WYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLV KGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSR

WQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 78 (,7C2 light chain):

DIVMTOSPDSLAVSLGERATINCRASQSVSGSRFTYMHWYQQKPGQ PPKLLIKYASILESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQH SWEIPPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLN NFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

ADYEKHKVYACEVTHOQGLSSPVTKSFNRGEC SEQ ID No. 79 ("7C2 heavy chain"):

EVOLVAOSGAEVKKPGASVKVSCKASGYSFTGYWMNWVRQAPGQAG LEWIGMIHPLDAEIRANQKFRDRVTITVDTSTSTAYLELSSLRSEDTAV YYCARGTYDGGFEYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGG TAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYSLSSV VTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSN KALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFY PSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ

GNVFSCSVMHEALHNHYTQKSLLSK SEQ ID No. 80 (,TZB-HC"*):

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKST SGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLOSSGLYS LSSVVTVPSSSLGTQTYICNVWNHKPSNTKVDKRVEPKSCDKTHTCPP CPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKF NWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYK CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCL VKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKS

RWQQGNVFSCSVMHEALHNHYTQKSLSLSK SEQ ID No. 81 (,TZB-LC*):

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLL IYSASFLYSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQHYTTP PTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPRE AKVOQWKVDNALQOSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKH

KVYACEVTHQGLSSPVTKSFNRGEC SEQ ID No. 82 ("GS-linker 1"): GGGGS SEQ ID No. 83 ("GS-linker 2"): SGGGSGGGGS SEQ ID No. 84 ("GS-linker 3"): GCGGGSGGGGSCGGGGES SEQ ID No. 85 ("GS -linker 4"): CGGGSGGGGSCGGGSCGGGES SEQ ID NO.86 ("GS-linker 5"): CGGGSGGGESCSCGGESGCGGESGGGES SEQ ID NO.87 ("GA-linker"): GAAGAAG SEQ ID NO.88 ("GT-linker"): GGTGGT SEQ ID NO. 89 ("ST-linker"): STISTS SEQ ID No. 90 ("PA-linker"): PAPAP SEQ ID No. 91 ("SA-linker"): SAASAAS SEQ ID No. 92 ("HF-oaFabFc light chain" / "A21-TZB-20a2 light chain"):

DIVLTQSPDSLAVSLGERATINCRSSQTLLYSNNQKNYLAWYQKKPG QPPKLLISWAFTRKSGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYC QQYSNYPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVKCL LNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGESDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNK ALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYP SDIAVEWESNGQPENNYDTTPPVLDSDGSFFLYSDLTVDKSRWQQG

NVFSCSVMHEALHNHYTQKSLSLSPGK SEQ ID No. 93 ("HF-oaFabFc heavy chain" / "A21-TZB-202 heavy chain"):

EVOLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLE WVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAV YYCSRWGGDGFYAMDYWGQGTLVTVSSGGGGSGCGGESCGGGESGÇE GGGSDIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGK APKLLIYSASFLYSGVPSRFSGSRSGTDFLTISSLQPEDFATYYCQQ HYTTPPTFGQGTKVEIKGSQVALVASGAEVKKPGASVKVSCKASGY SFTGYFINWVREAPGQGLEWMGHISSSYATSTYNQKFQGRVTFTVD TSSSTAYMELSSLRSEDTAVYYCVRSGNYEEYAMDYWGQGTLVTVS SASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGAL TSGVHTFPAVLOSSGLYSLESVVTVPSSSLGTQTYICNVNHKPSNTK VDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTP EVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVV SVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT LPPSRKEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LKSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSL

SPGK Example 1: Biparatopic anti-HER2 binding agents

[00271] The inventors have generated biparatopic IgG derivatives. Unlike other HER2-targeted biparatopic antibodies, eg Medimmune's antibody-drug conjugate (ADC) ME-DI4276 (Li et a/., 2016), these IgGs show very strong antitumor activity as binding proteins "naked", i.e. without bound drug (Kast et al., in preparation). Thus, it is believed that these

Biparatopic anti-HER2 IgGs combine the mechanisms of action of trastuzumab plus pertuzumab plus the action of small-molecule kinase inhibitors against HER2 into a single molecule. Furthermore, the potential off-target effects of biparatopic anti-HER2 IgGs are expected to remain far below those of ADC fusions such as T-DM1 or MEDI4276, as they can only act on HER2 “addicted” cells. while ADCs can, through their toxins, act on many healthy tissues. This opens the therapeutic windows for new combination therapies. Furthermore, inhibition of pan-ErbB by polymerization of HER2 receptors can passively block the compensatory activation of other receptor tyrosine kinases (RTKs). Biparatopic anti-HER2 binding agents interfere with the free lateral movement of HER2 receptors on the HER2-amplified cancer cell surface, but without inducing competent signaling complexes, which can block the activation of other RTKs. Consequently, biparatopic anti-HER2 binding agents may show strong synergies with small molecule inhibitors, which tend to induce the expression of compensatory RTKs that ultimately lead to therapy avoidance. Therefore, biparatopic anti-HER2 IgGs have a very high potential to elicit strong antitumor synergies in combination with small molecule inhibitors in a broad panel of HER2-amplified cancers. The potential for synergies with small molecule inhibitors is greater than current single specificity antibodies or antibody combinations.

[00272] Illustrative schematics of preferred biparatopic IgG constructs are shown in Fig. 1 and 2

[00273] Data regarding the preparation and biological activity of the biparatopic IgG constructs are shown in Figs 3 to 26. Protocol for the production of biparatopic IgGs in CHO cells

vector design

[00274] —Bicistronic plasmids containing two expression cassettes or two vector systems were constructed for co-transfections. Derivatives of plasmid pYMex10 (Morphosys) were used for the bicistronic strategy. In the resulting plasmid constructs, the coding sequences of the polypeptide chains of the multimeric constructs were each under the control of an individual CMV promoter and terminated by a polyA tail signal such as, for example, taken from bovine growth hormone or simian virus 40 (see Fig. 3 and 4). Vectors derived from pcDNA 3.1 (Thermo) were used for co-transfections. Here, the individual polypeptide chains are on a separate plasmid, reducing the risk of recombination of homologous elements and even allowing to adjust the plasmid molar ratios for transfections to improve yield. The exchange of genes of interest is possible through standard cloning techniques. Examples of the resulting constructs are represented in Fig. 5. Expression in CHO-S

[00275] Exponentially growing CHO-S cells (Thermo) were seeded in CHOgro (Mirrus) at a density of four million per ml in TPP600 bioreactors. Per ml of culture, 3 µg of linear polyethyleneimine (MW 25,000, PolySciences Inc) and 1.25 µg of highly pure plasmid DNA were added with intermediate mixing. Finally, cultures were supplemented with valproic acid to a final concentration of 1 MM. Proteins were expressed at 31º or 37 ºC, 8% CO2 and 180 mm with a 50 mm projection in a Kuhner ISF1-X shaker for up to 12 days. Molecule purification

[00276] Expression cultures were harvested by centrifugation at 1400 rpm and 4°C for 30 min. In addition, the supernatants fo-

were clarified by 0.22 µm filtration and adjusted to pH 7. All subsequent purification steps were carried out at 4°C. Supernatants were applied to PBS equilibrated rProtein A (GE) columns operated on an AEKTA Pure system. After washing with PBS, bound protein was eluted by 0.1 M glycine pH 2.75 (chromatogram Fig. 6). The fractions of interest were pooled and the buffer exchanged to 20 mM Bis/Tris methane 20 mM NaCl pH 6.75. Filtered protein solutions were loaded onto Resource S (GE) columns and eluted with a gradient from 100% Bis/Tris methane 20 nM NaCl to 1000 mM pH 6.75 (see Fig. 7). The desired fractions were pooled and, if necessary, polished on SEC (appropriately sized Superdex200 columns; GE) to high purity (Fig. 8). Monomeric fractions were pooled, filtered and purity analyzed on the SDS page (Fig. 9). Finally, pure protein was used for biochemical and cellular assays, as well as in vivo studies. HER2-related effects of tetravalent biparatopic IgGs

[00277] The tetrameric polypeptide constructs (tetravalent and biparatopic) 441 and 47C2 have been tested in several assays, all of which are well known to be versed, and compared to the MEDI4276 tetravalent IgG fusion (without a toxin), the dimeric bivalent bivalent polypeptide constructs 841, 87C2 and Fc fusions thereof, bivalent biparatopic DARPin construct 6L1G (see patent application WO 2014/060365 A1), unique IgGs (TZB, PZB, A21 and 7C2) and combinations thereof.

[00278] Growth inhibition was tested in an XTT cell viability assay using high-content live cell microscopy, Hoechst staining and cell counting. As shown in Table 1, constructs 441, 47C2, 841, 87C2 and the Fc fusions of 841 and 87C2, 6L1G and the combination of TZB with A21 resulted in total inhibition of growth, whereas single antibodies and TZB with-

combined with PZB lead to partial inhibition. Surprisingly, in the absence of a cytotoxic drug, the MEDI4276 antibody stimulated the growth of XTT cells.

[00279] The effect of the constructs on apoptosis/cell death was analyzed by high-content microscopy of live cells with annexin-V and PI staining or by detection of cleaved PARP in cell lysates by Western blot for analysis of PARP cleavage .

Constructs 441, 47C2, 841, 87C2 and their Fc, 6L1G fusions had an effect on apoptosis, whereas MEDI4276, single antibodies did not influence apoptosis, and TZB and A21 had a partial effect. The TZB+PZB combination is able to induce apoptosis in a very small fraction of cells or in fragile cell lines.

[00281] Cross-linking of HER2 on the cell surface, also called "blocking", was measured by fluorescence recovery after photodegradation (FRAP) and single-cell localization microscopy. A reduction in the FRAP signal indicates lower cell mobility and, therefore, cross-linking in response to the polypeptide constructs.

[00282] 441,47C2 and 6L1G resulted in blockade of receptors and the partial crosslinking effect was measured for 841, 87C2 and their Fc fusions, as well as TZB+PZB and TZB+A21 antibody combinations. Single antibodies had no effect on crosslinking.

[00283] In addition, the internalization of HER2 in cells was analyzed by a surface protein internalization and degradation assay, confocal microscopy and flow cytometry, as described in detail in example 2.

[00284] The tetravalent biparatopic constructs 441, 47C2 and ME-DI4276 exhibited a strong effect on the internalization of HER2, while an inhibition of recycling was detected for the combinations TZB+PZB and TZB+A21). The remaining constructs did not show ne-

no effect.

[0001] A The degradation of HER?2 was tested by a surface protein internalization and degradation assay and Western blot detection of total HER?2. Here, 441 and 47C2 lead to strong and rapid degradation. MEDI4276 had an effect on degradation, but less strong compared to 441 and 47C2. In contrast, the antibody combinations resulted in slow degradation, and the remaining constructs had no effect. Inhibition construct — of| Apoptosis/ |"Block"] |Internalization of |Degradation-growth |death ce-|(crosslinking |HER2 tion of squid in cells) HER2 441/47C2 yes Yes Yes Strong Fast, Strong MEDI4276 No (stimulus-|No No deter- |equal to 441 Less than (no toxin) |lant!) nated, but 441 expected 841/87C2 Yes Yes Partial No No Fc effusions of the same Antibodies Partial No No No No unique TZB+PZB Partial No (yes) | Crosslinking Inhibition of recycling (very weak) TZB+A21 Yes Partial Cross-linking Inhibition of recycling Table 1: Results of assays measuring the HER?2-related effects of polypeptide constructs

[00285] Table 2 shows the results of the binding studies to HER?2 performed with the same constructs as the experiments described above. Binding was determined by flow cytometry and additionally by size/scatter exclusion chromatography.

multi-angle light (SEC-MALS) in case of complex formation between the biparatopic IgG and HER2 constructs.

All constructs bind extracellular domains 1, 2 and/or 4 as expected (see Table 2). 441, 47C2 and the TZB + A21 combination exhibited an extremely slow dissociation rate that was slower compared to the remaining constructs.

[00287] Interestingly, all biparatopic constructs resulted in a HER2 binding stoichiometry of close to 1:1. Single antibodies and antibody combinations only lead to a stoichiometry of 1:2.

[0002] The serum half-life of the 441 construct was further tested by intravenous injection into NSG mice and time-resolved detection of biparatopic IgG in blood samples by ELISA. To determine the half-life of 441, 3 mg/kg of purified construct was injected intravenously into NSG mice. At the indicated time points, mice were bled, blood was allowed to clot, and serum was obtained by taking supernatants from centrifuged samples. A standard capture ELISA was used to assess serum 441 levels (Fig. 32). Mouse anti-human Fc antibody was coated directly onto maxisorb plates. Standards 441 bound and 441 with raised sera were released by alkaline phosphatase conjugated goat anti kappa chain antibodies. Data were fitted to a two-phase decay model and the resulting half-life was calculated to be 4.3 ± 45.3 hours (phases a and a).

1) ro

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[00288] Surprisingly, the tetravalent biparatopic tetrameric constructs 441 and 47C2 lead to strong inhibition of cell proliferation, induce cell death by apoptosis and lead to cross-linking of HER?2 on the cell surface and induce strong internalization of HER2 and strong degradation of HER2 , in addition to its excellent binding properties to HER2. 441 and 47C2 were superior or scored equally as well as all other constructs in all categories.

[00289] TZB+PZB and TZB+A21 resulted in a decrease in total HER2?2 (very weak in the case of TZB + PZB) which was attributed to the inhibition of recycling, a mechanism by which HER2 is degraded without previous intracellular accumulation (See Fig. 35).

To determine the effect of construct 441 on tumor growth, beige SCID mice (Charles River) were inoculated on the right flank with five million N87 cells in 50% matrigel (Corning). After the tumors reached about 150 mm?, the mice were treated with 441 10 mg/kg eight times with an interval of three to four days. Treated mice responded to 441 with reduced tumor burden. Growth arrest was initially observed for mice treated with TZB (10 mg/kg) and hA21G (10 mg/kg) and control mouse tumors (labeled 'PBS' in Fig. 33) showed unimpeded progression (Fig. 33).

[00291] "Example 2: Enhanced internalization, lysosomal trafficking and degradation of HER2 by the disclosed molecule 441

[00292] — Microscopy with BT-474 and HCC1419 breast cancer cells

[00293] For microscopy of fixed samples, cells were seeded at a density of 4-10º cm”? in pu slides (Ibidi, cat. No. 80824) in complete medium. The next day, cells were treated with the respective molecules. After 2 hours, cells were washed once with Dulbecco's phosphate-buffered saline (DPBS) and fixed by adding 4% (w/v) paraformaldehyde dissolved in DPBS and incubating at room temperature for 10 min. Then, cells were washed twice with PBSBA+T (DPBS supplemented with 1% (w/v) bovine serum albumin (BSA), 0.1% (w/v) sodium azide and 0.5% (w/v) Tween-20). Subsequently, the cells were incubated in anti-LAMP antibody (Cell Signaling Technology, cat. No. D401S) dissolved at 1:150 (v/v) in PBSBA+T, supplemented with 100 ng ml"* 2-(4- amidinophenyl)-1H-indole-6-carboximidamide (DAPI) for 30 min at room temperature.The cells were then washed twice with PBSBA+T and, subsequently, anti-mouse antibodies, conjugated to Alexa Fluor 488 (Thermo Fis - her Scientific, cat. No. A11001) and goat anti-human, conjugated to Alexa Fluor 647 (Thermo Fisher Scientific, cat. No. A-21445), dissolved in PBSBA+T, were added and incubated for 30 min at room temperature. Then, the cells were washed twice with PBSBA+T, fixed once more by adding 4% paraformaldehyde (w/v) dissolved in DPBS and incubating at room temperature for 10 min. finally washed once with PBSA and stored in PBSA (DPBS supplemented with 0.1% sodium azide (w/v)) at 4°C until measurement.The image was performed on a microscope the SP5 laser scanning confocal (Leica). The images show that, for both cell lines, only 441 were able to induce their rapid internalization and show strong colocalization with lysosomal compartments (LAMP1 positive).

Surface protein internalization and degradation assay

[00294] To quantify the internalization and degradation of HER2 after treatments, we performed a quantitative assay of internalization and degradation of surface proteins. In summary, a stable HEK293 Flp-ln TREx cell line (Thermo Fisher Scientific, cat.

K650001) was generated according to the manufacturer's instructions, in which a HaloTag-HER2 receptor fusion can be overexpressed after induction. For the assay, cells were seeded two days before the first treatment, and one day before treatment, doxycycline was added to induce stable overexpression for 24 hours. Treatments (100 nM) were added at indicated time points, referring to the time of cell labeling.

[00295] Upon completion of the treatment time intervals, cells were tagged in a two-step procedure. A HaloTag ligand containing Alexa Fluor 660 (HTL-AF660, Promega, cat. No. G8472), which is completely impervious to cells and therefore stains only surface receptors, was coupled in a first labeling step. A cell-permeable HaloTag ligand containing tetramethyl rhodamine (HTL-TMR, Promega, cat. No. G8252) was, in the second step, applied to stain the entire receptor fusion, which resides in the intracellular compartments. Thus, the signals coming from the surface and the internal receiver are detected in separate channels in a flow cytometer. Therefore, information on the location and, using the rescheduling procedure described below, on the quantitative distribution can be obtained. A commercially available dead cell stain was used to exclude permeabilized (dead) cells from the analysis, for which all receptors would appear to be on the surface. Fluorescence intensities in each channel for 2000-10,000 cells were recorded using a LSR Il Fortessa (BD) and blocked non-permeabilized single cells. The mean fluorescence intensities of these populations were obtained using FlowJo 10.4 (FlowJo). Data processing

[00296] To correct the different detection efficiencies of the flow cytometry instrument in the channels for AF660 and TMR, the

have been scaled, producing relative abundances. A control sample (utr., s.), in which the first step (marking with HTL-AF660) is omitted, is required for this purpose. In this sample, all HaloTag molecules will react with HTL-TMR in this “unique” (s.) tagging procedure, regardless of their location. Using the mean fluorescence intensities (MFI) of the non-permeabilized singlet cell population, the normalized Stmr signal (in characteristic scale) in the TMR channel for a sample is obtained by normalization for an untreated control sample. with single markup (utr.,s.) and background subtraction: where utr.,, unlab. represents an untreated, unlabeled control (showing only autofluorescence).

[00297] The first step of surface marking with cell impermeable dye is virtually saturating in the complete two-step (double) marking procedure. The Sareso normalized surface signal can thus be defined in:

[00298] The signal from a sample can be related to the surface signal of a dual labeled control (utr., d.) and does not require a separate sample with single staining, however, because the internal receiver is not accessible for HTL- AF660 and therefore cannot be stained.

[00299] —AStvmR, the difference of the single labeling procedure to the double labeling procedure in the TMR channel, now corresponds exactly to the number of molecules that were blocked by the first surface specific step. The signal in the AF660 channel in the same double tagging experiment is also a direct correlation of this number of molecules:

ASTwa = Str (Utr.,$.) — Srug (Utr., 0.) = Sazg3o, dmensicnade(Utr.,d.) — (eq.S51).

[00300] A correction factor Ca can thus be defined, which relates the measured intensity Sareso(utr.d.) (recorded in channel AF660) to Sarse6o,scaled(Utr.,d.) (in the scale of TMR channel): SaFego, sized CULT .,d.) = Sarggo Cult ..d.) x Ca (eq.S1).

[00301] Using single tag and double tag untreated cell signals, calculation of Ca is possible as follows: n= Sriar(utr.,s.) — Sus (utr.,0.) — 290% — Srvrs Cutr.,d.) Geq. 51), Sarego (utr., d.) Sareso (utr., d.)

[00302] taking into account that the TMR signal from untreated cells with unique labeling was, using eq. S1, scaled to 100% before. The correction of the signals registered in the AF660 channel can now be done according to: Sareso, dimensioned (18 113 = Ca x Sargenlre dd) (en 51) for the treated samples.

[00303] *“Srmr(tre., d.) and Sareso, sized(tre., d.) then truly represent the abundance of internal and surface proteins, respectively, and the sum Sar6ço, sized(tre.,d. )+SrmRr(tre.,d.) represents the amount of total protein, for a double-labeled treated sample, always relative to an untreated control sample.

[00304] The data sizing as described above was performed and the results were plotted using MATLAB R2017b (MathWorks), R 3.5.1, Prism 6.07 (GraphPad) and Excel2016 (Microsoft). The results are shown in Fig. 35. Unlike all other constructs, construct 441 showed near internalization of HER2 after less than 5 minutes.

Claims (15)

1. Tetrameric polypeptide, characterized in that it comprises or consists of a. a first polypeptide chain comprising a first VL antigen binding domain and a first constant CL domain, b. a second polypeptide chain comprising a first VH antigen binding domain, a first CH1 constant domain, a first CH2 constant domain, and a first CH3 constant domain, c. a first linker that specifically binds to a D4 epitope of HER2, wherein said first linker is comprised on said first polypeptide chain and linked to the N-terminus of said first VL antigen binding domain by a first amino acid linker of interdomain, or said first linker is comprised on said second polypeptide chain and linked to the N-terminus of said first VH antigen-binding domain by a first interdomain amino acid linker, d. wherein the first VL antigen-binding domain of the first polypeptide chain and the first VH antigen-binding domain of the second polypeptide chain together constitute a second linker, particularly a Fab domain, that binds specifically to a D1 epitope of HER?2, e.g. a third polypeptide chain comprising a second VL antigen binding domain and a second CL constant domain, f. a fourth polypeptide chain comprising a second VH antigen-binding domain, a second CH1 constant domain, a second CH2 constant domain, and a second CH3 constant domain, g. a third linker that specifically binds to a D4 epitope of HER2, wherein said third linker is comprised within said third polypeptide chain and linked to the N-terminus of said second VL antigen-binding domain by a second linker of interdomain amino acid, or said third linker is comprised within said fourth polypeptide chain and linked to the N-terminus of said second VH antigen-binding domain by a second interdomain amino acid linker, h. wherein the second VL antigen-binding domain of the third polypeptide chain and the second VH antigen-binding domain of the fourth polypeptide chain together constitute a fourth linker, particularly a Fab domain, that specifically binds to a D1 epitope of HER?2, 2. Polypeptide according to claim 1, characterized in that - said first polypeptide chain and said third polypeptide chain and/or said second polypeptide chain and said fourth polypeptide chain have characteristics of a sequence identity to each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, wherein more particularly said first polypeptide chain and said third polypeptide chain and /or said second polypeptide chain and said fourth polypeptide chain are identical and/or wherein - said first linker and said third linker have characteristics of a sequence identity with each other of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, wherein more particularly said first binder and said third binder are identical. 3. Tetrameric polypeptide according to any one of the preceding claims, characterized in that said first CH2 constant domain and said first CH3 constant domain of said second polypeptide chain interact with, particularly are covalently linked with, said second CH2 constant domain and said second CH3 constant domain of said fourth polypeptide chain, such that a tetrameric polypeptide is formed. 4. Tetrameric polypeptide according to any one of the preceding claims, characterized in that said second polypeptide chain comprises a first hinge region between said first constant domain CH1 and said first constant domain CH2, and said fourth polypeptide chain comprises a second hinge region between said second CH1 constant and said second CH2 constant domain, wherein said first hinge region and said second hinge region mediate complex formation between the said second polypeptide chain and said fourth polypeptide chain, particularly by at least one disulfide bond, such that a tetrameric polypeptide is formed. 5. Polypeptide, according to any of the preceding claims, characterized in that said first linker and/or said third linker comprises or consists of a single chain variable fragment polypeptide chain comprising giving an scFv heavy chain, a scFv linker chain and an scFv light chain, wherein particularly a. said scFv heavy chain of said first linker and/or said third linker comprises or consists of a peptide sequence with a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more , even more particularly 95% or more, with a peptide sequence. deo selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54 and SEQ ID NO.80, in which more particularly the said scFv heavy chain of the first linker and/or said third linker comprises or consists of a peptide sequence identical to a peptide sequence selected from SEQ ID NO.15, SEQ ID NO.21, SEQ ID NO. 22, SEQ ID NO.23, SEQ ID NO.44, SEQ ID NO.53, SEQ ID NO.54 and SEQ ID NO.80, and b. said scFv light chain of said first linker and/or said third linker comprises a peptide sequence has characteristics of a sequence identity of 70% or more, more particularly 80% or more, even more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.14, SEQ ID NO.24, SEQ ID NO.25, SEQ ID NO.26, SEQ ID NO.43 and SEQ ID NO.81, in that more particularly said scFv light chain of said first linker and/or said third linker comprises a peptide sequence identical to a peptide sequence selected from SEQ ID No. 14, SEQ ID No. 24, SEQ ID No. 25, SEQ ID No. 26, SEQ ID No. 43 and SEQ ID No. 81. 6. Polypeptide according to claim 5, characterized in that the scFv linker chain comprises a peptide sequence has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 16, wherein more particularly said scFv linker chain comprises a peptide sequence identical to SEQ ID No. 16. 7. Polypeptide, according to any of the preceding claims, characterized by the fact that a. said first polypeptide chain and/or said third polypeptide chain comprising a peptide sequence has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO.32, SEQ ID NO.39, SEQ ID NO.41, SEQ ID NO. 50 and SEOQ ID. 76, wherein more particularly said first polypeptide chain and/or said third polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID NO.18, SEQ ID NO.30, SEQ ID NO.31, SEQ ID No. 32, SEQ ID No. 39, SEQ ID No. 41, SEQ ID No. 50 and SEQ ID No. 76, and b. said second polypeptide chain and/or the fourth polypeptide chain comprises a peptide sequence has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more - particularly 95% or more, with a peptide sequence selected from SEQ ID NO.19, SEQ ID NO.20, SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID No. 42, SEQ ID No. 51, SEQ ID No. 52 and SEQ ID No. 52. 77, wherein more particularly said second polypeptide chain and/or said fourth polypeptide chain comprises a peptide sequence identical to a peptide sequence selected from SEQ ID No. 19, SEQ ID No. 20, SEQ ID No. 27 , SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.40, SEQ ID NO.42, SEQ ID NO.51, SEQ ID NO.52, and SEQ ID NO. 77. 8. Polypeptide, according to any of the preceding claims, characterized by the fact that a. said first polypeptide chain and/or said fourth polypeptide chain comprises a peptide sequence has characteristics of a sequence identity of 70% or more, particularly. largely 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No. 36, SEQ ID No. 37, SEQ ID No. 38 and SEQ ID No. 78, wherein more particularly said first strand polypeptide and/or said fourth polypeptide chain comprises a peptide sequence identical to SEQ ID No. 36, SEQ ID No. 37, SEQID No. 38 and SEQ ID No. 78, and b. said second polypeptide chain and/or said fourth polypeptide chain comprising a peptide sequence has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or more, with SEQ ID No.33, SEQ ID No.34, SEQ ID No.35 and SEQ ID No.79, wherein more particularly said second polypeptide chain and/or said fourth polypeptide chain comprises a sequence of peptide identical to SEQ ID NO.33, SEQ ID NO.34, SEQ ID NO.35 and SEQ ID NO.79. 9. Polypeptide, according to any of the preceding claims, characterized by the fact that a. said first polypeptide chain and/or said third polypeptide chain is has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or further, with SEQ ID No. 1, wherein more particularly said first polypeptide chain and/or said third polypeptide chain is identical to SEQ ID No. 1, and b. said second polypeptide chain and/or said fourth polypeptide chain is has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or further, with SEQ ID No. 2, wherein more particularly said second polypeptide chain and/or said fourth polypeptide chain is identical to SEQ ID No. 2. 10. Polypeptide, according to any of the preceding claims, characterized by the fact that a. said first polypeptide chain and/or said third polypeptide chain is has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or further, with SEQ ID No.3 wherein more particularly said first polypeptide chain and/or said third polypeptide chain is identical to SEQ ID No.3, and b. said second polypeptide chain and/or said fourth polypeptide chain is has characteristics of a sequence identity of 70% or more, particularly 80% or more, more particularly 90% or more, even more particularly 95% or further, with SEQ ID No. 4, wherein more particularly said second polypeptide chain and/or said fourth polypeptide chain is identical to SEQ ID No. 4. 11. Polypeptide according to any one of the preceding claims, characterized in that said first interdomain amino acid linker and/or said second interdomain amino acid linker consists of 1, 2, 3, 4 , 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids, and wherein said first interdomain amino acid linker and/or said second interdomain amino acid linker comprises or consists of amino acids G, A, J, S, T, P, C, V, Me E, particularly wherein said first interdomain amino acid linker is /or said second interdomain amino acid linker comprises or consists of amino acids G, S, A and T, more particularly wherein said first interdomain amino acid linker and/or said second amino acid linker of interdomain is characterized by an amino acid sequence. dos (GGGGS)n, with n being 1, 2, 3, 4 or 5 or said first interdomain amino acid linker and/or said second interdomain amino acid linker comprises or consists of a peptide sequence selected to from one of SEQ ID NO.17, SEQ ID NO.55 to SEQ ID NO.69 and SEQ ID NO.82 to SEQ ID NO.91 or a functional equivalent peptide sequence has sequence identity characteristics of at least 70%. 12. Polypeptide, according to any one of claims 1 to 11, characterized by the fact that it is for use in a method for the prevention or treatment of a malignant neoplastic disease associated with the expression of HER?2. 13. Isolated nucleic acid, characterized in that it encodes at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the tetrameric polypeptide as defined in any one of claims 1 to 11 . 14. A host cell that is adapted to produce at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and the fourth polypeptide chain of the polypeptide as defined in any one of claims 1 to 11, characterized in by the fact that the host cell particularly comprises the isolated nucleic acid as defined in claim 13, so that the host cell is capable of producing at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain and fourth polypeptide chain of the polypeptide as defined in any one of claims 1 to 11. 15. Method for obtaining the polypeptide as defined in any one of claims 1 to 11, characterized in that the method comprises culturing the host cell as defined. according to claim 14, such that at least one of the first polypeptide chain, the second polypeptide chain, the third polypeptide chain, and the fourth polypeptide chain of the polypeptide, as defined in any one of claims 1 to 11, is produced.