Her2 binding immunoglobulin single variable domains, constructs comprising the same and uses thereof

Abstract

The present technology provides HER2-binding immunoglobulin single variable domains (ISVDs), polypeptides comprising such HER2-binding ISVDs, and polypeptide constructs comprising such HER2- binding ISVDs and an Fc region. The present technology further provides nucleic acids encoding said ISVDs, polypeptides, and polypeptide constructs as well as vectors, hosts and methods to produce these ISVDs, polypeptides, and polypeptide constructs. Moreover, the present technology relates to methods of treatment making use of the ISVDs, polypeptides or polypeptide constructs according to the present technology.

Description

[0001] HER2 BI NDING IMM UNOGLOBULIN SI NGLE VARIABLE DOMAINS, CONSTRUCTS

[0002] COMPRISI NG THE SAM E AND USES THEREOF

[0003] FI ELD OF TH E PRESENT TECH NOLOGY

[0004] The present technology provides HER2-binding immunoglobulin single variable domains (ISVDs), polypeptides comprising HER2-binding ISVDs, and polypeptide constructs comprising HER2-binding ISVDs and an Fc region. The present technology further provides nucleic acids encoding said ISVDs, polypeptides, and polypeptide constructs as well as vectors, hosts and methods to produce these ISVDs, polypeptides, and polypeptide constructs. Moreover, the present technology relates to methods of treatment making use of the ISVDs, polypeptides or polypeptide constructs according to the present technology.

[0005] TECHNOLOGICAL BACKGROU ND

[0006] Cancer takes an enormous human toll around the world. It is nowadays the second leading cause of death globally, only preceded by heart disease and stroke. Cancers figure among the leading causes of morbidity and mortality worldwide, with approximately 19.3 million new cases and 10 million cancer related deaths in 2020. The number of new cases is expected to rise further over the next decades. Population growth, ageing and lifestyle changes have been described as contributing factors to the increasing cancer burden. In 2013, the WHO projected that by 2030 cancer will surpass ischemic heart disease and become the most common cause of death worldwide (source: WHO Cancer).

[0007] The total economic impact of premature death and disability from cancer worldwide was already about $900 billion in 2008, which represented 1.5% of the world's gross domestic product at that time. With cancer becoming increasingly common, the total economic impact is sure to have increased significantly as well. Available treatment regimens for solid tumors typically include a combination of surgical resection, chemotherapy, and radiotherapy. In 40 years of clinical experience little progress has been achieved, especially in advanced stages of cancer. New therapies combatting cancer are eagerly awaited. Antibody therapy is now an important part of the physician's armamentarium to battle diseases and especially cancer. Monoclonal antibodies have been established as a key therapeutic approach for a range of diseases already for several years.

[0008] Different types of antibody therapeutics have become available since the first discovery of monoclonal antibodies, including bispecific antibodies, single domain antibodies, antibody fragments, fusion proteins, and more.

[0009] More recently, immunotherapy has emerged as a rapidly growing area of cancer research. Immunotherapy is directing the body's immune surveillance system, and in particular T cells, to cancer cells.

[0010] Receptor tyrosine-protein kinase erbB-2, more commonly referred to in humans as HER2, is a protein that normally resides in the membranes of cells and is encoded by the ERBB2 gene. H ER2 is a member of the human epidermal growth factor family, which when overexpressed or amplified, has shown to play an important role in the development and progression of certain types of cancer. HER2 overexpression is known to occur in breast, ovarian, stomach, uterine as well as gastric cancer. Consequently, HER2 has become an important biomarker as well as target for cancer immunotherapies.

[0011] The most well-known and successful anti-HER2 immunotherapy is trastuzumab (brand name Herceptin®). Trastuzumab is a monoclonal antibody that targets HER2 and is recommended for all patients with HER2- positive breast cancer who are also receiving chemotherapy.

[0012] Another known anti-HER2 therapy is pertuzumab (brand name Perjeta®). This monoclonal antibody is indicated as a combination therapy with trastuzumab and docetaxel for the treatment of metastatic HER- 2 positive breast cancer.

[0013] Both therapies do have downsides. Firstly, since they are both combined with chemotherapy, there are many negative side effects for the patients. In addition to this, trastuzumab has had significant attention over its negative cardiac effects, while pertuzumab has some studies that show it has no "added benefit" when used in concurrence with trastuzumab and chemotherapy. Furthermore, since these therapies have only been approved for HER2-positive breast cancer, other HER2- positive cancers currently do not have a HER2-specific treatment option.

[0014] A need for improved HER2-targeting immunotherapies therefore still exists.

[0015] SUMMARY OF THE PRESENT TECH NOLOGY

[0016] The present technology relates to HER2-binding ISVDs and constructs comprising such HER2-binding ISVDs as well as uses thereof.

[0017] The present inventors have found novel HER2-binding ISVDs that are capable of specifically targeting and binding HER2. Interestingly, these HER2-binding ISVDs do not bind to the same epitope as the standard of care monoclonal antibody therapies trastuzumab and pertuzumab. These ISVDs are interesting candidates for use in both first-line treatment against HER2-positive tumors as well as a possible combination treatment with either trastuzumab or pertuzumab.

[0018] Therefore, in a first aspect, the present technology relates to an immunoglobulin single variable domain (ISVD) which specifically binds to human and / or non-human primate HER2, wherein said ISVD essentially consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO:1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XsIXeRVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein Xi is selected from I and S;

[0019] X2is selected from V, R and Y;

[0020] X3is selected from A and R;

[0021] X4is selected from I, L and M;

[0022] X5is selected from G and L;

[0023] X6is selected from D and S;

[0024] X7is selected from E and G;

[0025] X8is selected from D, E and V; or e. the amino acid sequence of CDR1 is X9IPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIXi2SXi3GXi4PXi5 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is VNG DXM (SEQ ID NO: 254), according to AbM definition, wherein

[0026] Xi is selected from I or S;

[0027] X2is selected from V, R or Y;

[0028] X3is selected from A or R;

[0029] X4is selected from I, L or M;

[0030] X5is selected from G or L;

[0031] X6is selected from D or S;

[0032] X7is selected from E or G;

[0033] Xg is selected from D, E or V;

[0034] X9is selected from G or R;

[0035] Xio is selected from F or A;

[0036] Xu is selected from T or I;

[0037] Xi2is selected from R or G;

[0038] Xi3is selected from absence of an amino acid residue or S;

[0039] X14 is selected from A or T;

[0040] Xis is selected from V or A;

[0041] Xie is selected from I or Y; f. the amino acid sequence of CDR1 is X9IPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TXi7Xi2SXi3GXi4PXi5 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is VNGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0042] X3is selected from A and R;

[0043] X4is selected from I, L and M; X9is selected from G and R;

[0044] Xio is selected from F and A;

[0045] Xu is selected from T and I;

[0046] X12 is selected from R and G;

[0047] X13 is selected from absence of an amino acid residue or S;

[0048] X14 is selected from A and T;

[0049] X15 is selected from V, Y and A;

[0050] Xi6 is selected from I and Y;

[0051] X17 is selected from I and S; and wherein the full amino acid sequence of the ISVD is different from SEQ ID NOs: 29-32.

[0052] In another aspect, the present technology relates to a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR sequences selected from the following: a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO: 1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XSIX6RVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein

[0053] Xi is selected from I and S;

[0054] X2is selected from V, R and Y;

[0055] X3is selected from A and R;

[0056] X4is selected from I, L and M; X5is selected from G and L;

[0057] X6is selected from D and S;

[0058] X7is selected from E and G;

[0059] Xg is selected from D, E and V; or e. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIX12SX13GX14PX15 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is VNG DXM (SEQ ID NO: 254), according to AbM definition, wherein

[0060] Xi is selected from I or S;

[0061] X2is selected from V, R or Y;

[0062] X3is selected from A or R;

[0063] X4is selected from I, L or M;

[0064] X5is selected from G or L;

[0065] X6is selected from D or S;

[0066] X7is selected from E or G;

[0067] Xg is selected from D, E or V;

[0068] X9is selected from G or R;

[0069] X10 is selected from F or A;

[0070] Xu is selected from T or I;

[0071] X12 is selected from R or G;

[0072] Xi3is selected from absence of an amino acid residue or S;

[0073] X14 is selected from A or T;

[0074] X15 is selected from V or A:

[0075] Xie is selected from I or Y; f. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TXi7Xi2SXi3GXi4PXi5 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is VNGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0076] X3is selected from A and R;

[0077] X4is selected from I, L and M;

[0078] X9is selected from G and R;

[0079] X10 is selected from F and A;

[0080] Xu is selected from T and I;

[0081] X12 is selected from R and G; X13 is selected from absence of an amino acid residue or S;

[0082] X14 is selected from A and T;

[0083] X15 is selected from V, Y and A;

[0084] Xi6 is selected from I and Y;

[0085] X17 is selected from I and S; and wherein the second ISVD has a sequence that may be the same or different from the first ISVD.

[0086] Additionally, when used in a polypeptide in combination with a T cell engaging ISVD, the HER2-binding ISVDs of the present technology have desirable binding affinities for specifically targeting HER2, allowing the T cells to be redirected to only tumor cells that are HER2-positive, resulting in specific tumor killing.

[0087] Furthermore, the inventors found that by combining the HER2-binding ISVDs of the present technology not only with a T cell engaging ISVD, but also with an Fc region, similar tumor cell killings were observed resulting in some even more potent molecules.

[0088] Consequently, in a further aspect, the present technology relates to a polypeptide construct comprising

[0089] (i) a polypeptide according to the present technology (specifically binding to human and / or nonhuman primate HER2);

[0090] (ii) a further (e.g., a second or a third) ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell; and

[0091] (iii) an Fc region which is linked to the polypeptide via a suitable linker or hinge region.

[0092] The present technology also relates to methods to produce such ISVDs, polypeptides and polypeptide constructs, as well as uses of such ISVDs, polypeptides and polypeptide constructs.

[0093] BRI EF DESCRIPTION OF DRAWINGS

[0094] Figure 1 shows the binding by the HER2 binding ISVDs according to the present technology to JIMT-1 (1A) and SKBR-3 (IB) cells as determined in flow cytometry (FACS). Figure 2 shows dose response curves of the TCE-HER2-ALB constructs in the impedance-based human T cell mediated MCF-7 (2A) and HCC1954 (2B) cell TDCC assay using an effector to target ratio of 8 to 1 in the presence of 30 pM HSA.

[0095] Figure 3 shows the residues within HER2 at a distance of 4 (3A, 3C, 3E, 3G) or 5 (3B, 3D, 3F, 3H) A from 47D05 (3A and 3B), 27A05 (3C and 3D), 29E09 (3E and 3F) and 27E07 (3G and 3H) as determined by Cryo- EM.

[0096] Figure 4 shows the structure model of HER2 in complex with ISVD 27E07, 47D05 and 27A05 and either with trastuzumab (4A) or with trastuzumab and pertuzumab (4B).

[0097] Figure 5 shows schematically the formats ISVD-only (5A) and ISVD-Fc (5B) (created with Biorender).

[0098] Figure 6 shows dose response curves of the bivalent HER2 binding ISVD constructs in the impedance-based human T cell mediated ZR-75-1 (6A) and HCM (6B) cell TDCC assay using an effector to target ratio of 8 to 1 in the presence of 30 pM HSA.

[0099] Figure 7 shows dose response curves of the bivalent HER2 binding ISVD constructs in the Incucyte-based ZR-75-1 proliferation assay in the presence of 30 pM HSA.

[0100] Figure 8 shows dose response curves of the bivalent HER2 binding ISVDs as T cell engagers in the format of both ISVD-only (8A and 8B) and ISVD-Fc (8C and 8D) in the impedance-based human T cell mediated ZR-75-1 (8A and 8C) and HCM (8B and 8D) cell TDCC assay using an effector to target ratio of 5 to 1 in the presence of 30 pM HSA.

[0101] Figure 9 shows dose response curves of the bivalent HER2 binding ISVD constructs in the impedance-based human T cell mediated ZR-75-1 cell TDCC assay using an effector to target ratio of 5 to 1 in the presence of 30 pM HSA.

[0102] Figure 10 shows dose response curves of the bivalent HER2 binding ISVD constructs in the Incucyte-based ZR-75-1 cell proliferation assay in the presence of 30 pM HSA. Figure 11 shows dose response curves of the T cell engaging bivalent HER2 binding ISVD constructs in the format of both ISVD-only and ISVD-Fc in the Incucyte-based human T cell mediated BT20 spheroids TDCC assay using an effector to target ratio of 1 to 5 in the presence of 30 pM HSA.

[0103] Figure 12 shows the geometric means of the IC50 values in the impedance-based TDCC assay (TDCC) and of the EC50 values in the FACS-based T cell activation assay for ISVD constructs in the format of both ISVD- only and ISVD-Fc on the cell lines ZR-75-1 (12A) and BT20 (12B).

[0104] Figure 13 shows the geometric means of the IC50 values in the impedance-based TDCC assay (TDCC) and of the EC50 values in the cytokine release assay for ISVD constructs in the format of both ISVD-only and ISVD-Fc on the cell lines ZR-75-1 (13A) and BT20 (13B).

[0105] Figure 14 shows the tumor volume (Median + / - error) in mm3by treatment group for A032801773 in ZR- 75-1 tumor bearing NOG mice engrafted with in vitro expanded T cells.

[0106] Figure 15 shows the tumor volume (Median + / - error) in mm3by treatment group for TPP-45142 in ZR-75- 1 tumor bearing NOG mice engrafted with in vitro expanded T cells -Tumor volume changes from baseline in mm3: Median (nMAD), n and p-value.

[0107] Figure 16 shows exposure of the89Zr labelled ISVD molecules in plasma overtime in ZR-75-1 tumor bearing NSG mice engrafted with in vitro expanded T cells.

[0108] Figure 17 shows tumor accumulation of the89Zr labelled ISVD molecules in ZR-75-1 tumor bearing NSG mice engrafted with in vitro expanded T cells.

[0109] Figure 18 shows dose response curves of A032801773 and TPP-45142 in the impedance-based human T cell mediated human primary cells TDCC assay using an effector to target ratio of 5 to 1 in the presence of 30 pM HSA, and one representative T cell donor (D1777). The human primary cells used were human cardiac myocytes (18A), human bronchial epithelia cells (18B) and primary human hepatocytes (18C) in comparison to the cancer cell line BT-549 (18D), all expressing the same levels of HER2 receptors per cell (5-10k HER2 / cell). T017001014 and TPP-45161 are the negative controls (no HER2 binding) for A032801773 and TPP-45142, respectively. Figure 19 shows super resolution confocal microscopy images and the frequency distribution of HER2 enriched areas on BT-549 cancer cells (19A) and on cardiac myocytes (19B).

[0110] Figure 20 shows the fold change in cytokine levels induced by A032801773 (black bar) and TPP-45142 (white bar) compared to the respective negative controls, T0177001014 and TPP-45161, at 3 different concentrations in the MIMIC" based Cytokine Release Assay (CRA) platform. The cytokines shown are: I FNg (20A), IL-6 (20B), TNFa (20C), IL- 10 (20D), IL-2 (20E) and GM-CSF (20F). Error bars represent 95% Cl: *** Adjusted p-value <0.0001; ** Adjusted p-value <0.002; * Adjusted p-value <0.02.

[0111] DETAILED DESCRIPTION

[0112] Unless indicated or defined otherwise, all terms used have their usual meaning in the art, which will be clear to the skilled person. Reference is for example made to the standard handbooks such as Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual (2nd Ed.) Vols. 1-3, Cold Spring Harbor Laboratory Press), F. Ausubel et al. (1987, Current protocols in molecular biology, Green Publishing and Wiley Interscience, New York), Lewin (1985, Genes II, John Wiley & Sons, New York, N.Y.), Old et al. (1981, Principles of Gene Manipulation: An Introduction to Genetic Engineering (2nd Ed.) University of California Press, Berkeley, CA), Roitt et al. (2001, Immunology (6th Ed.) Mosby / Elsevier, Edinburgh), Roitt et al. (2001, Roitt's Essential Immunology (10th Ed.) Blackwell Publishing, UK), and Janeway et al. (2005, Immunobiology (6th Ed.) Garland Science Publishing / Churchill Livingstone, New York), as well as to the general background art cited herein.

[0113] Unless indicated otherwise, all methods, steps, techniques and manipulations that are not specifically described in detail can be performed and have been performed in a manner known per se, as will be clear to the skilled person. Reference is for example again made to the standard handbooks and the general background art mentioned herein and to the further references cited therein; as well as to for example the following reviews; Presta (2006, Adv. Drug Deliv. Rev. 58 (5-6): 640-56), Levin and Weiss (2006, Mol. Biosyst. 2(1): 49-57), Irving et al. (2001, J. Immunol. Methods 248(1-2): 31-45), Schmitz et al. (2000, Placenta 21 Suppl. A: S106-12), Gonzales et al. (2005, Tumor Biol. 26(1): 31-43), which describe techniques for protein engineering, such as affinity maturation and other techniques for improving the specificity and other desired properties of proteins such as immunoglobulins. The term "sequence" as used herein (for example in terms like "immunoglobulin sequence", "antibody sequence", "variable domain sequence", "VHH sequence" or "protein sequence"), should generally be understood to include both the relevant amino acid sequence as well as nucleic acids or nucleotide sequences encoding the same, unless the context requires a more limited interpretation.

[0114] "Amino acid sequences" are interpreted to mean a single amino acid or an unbranched sequence of two or more amino acids, depending on the context. Nucleotide sequences are interpreted to mean an unbranched sequence of 3 or more nucleotides.

[0115] Amino acids are those L-amino acids commonly found in naturally occurring proteins and are listed in Table A-l. Those amino acid sequences containing D-amino acids are not intended to be embraced by this definition. Any amino acid sequence that contains post-translationally modified amino acids may be described as the amino acid sequence that is initially translated using the symbols shown in the Table A-l with the modified positions; e.g., hydroxylations or glycosylations, but these modifications shall not be shown explicitly in the amino acid sequence. Any peptide or protein that can be expressed as a sequence modified linkages, cross links and end caps, non-peptidyl bonds, etc., is embraced by this definition.

[0116] The terms "protein", "peptide", "protein / peptide", and "polypeptide" are used interchangeably throughout the disclosure, and each has the same meaning for purposes of this disclosure. Each term refers to an organic compound made of a linear chain of two or more amino acids. The compound may have ten or more amino acids; twenty-five or more amino acids; fifty or more amino acids; one hundred or more amino acids, two hundred or more amino acids, and even three hundred or more amino acids. The skilled artisan will appreciate that polypeptides generally comprise fewer amino acids than proteins, although there is no art-recognized cut-off point of the number of amino acids that distinguish a polypeptide from a protein; that polypeptides may be made by chemical synthesis or recombinant methods; and that proteins are generally made in vitro or in vivo by recombinant methods as known in the art.

[0117] Unless the context clearly requires otherwise, throughout the description and the claims, the words "comprise", "comprising", and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of "including, but not limited to". For instance, when a nucleotide sequence, amino acid sequence or polypeptide is said to "comprise" another nucleotide sequence, amino acid sequence or polypeptide, respectively, or to "essentially consist of" another nucleotide sequence, amino acid sequence or polypeptide, this may mean that the latter nucleotide sequence, amino acid sequence or polypeptide has been incorporated into the first mentioned nucleotide sequence, amino acid sequence or polypeptide, respectively, but more usually this generally means that the first mentioned nucleotide sequence, amino acid sequence or polypeptide comprises within its sequence a stretch of nucleotides or amino acid residues, respectively, that has the same nucleotide sequence or amino acid sequence, respectively, as the latter sequence, irrespective of how the first mentioned sequence has actually been generated or obtained (which may for example be by any suitable method described herein). By means of a non-limiting example, when a polypeptide of the present technology is said to comprise an immunoglobulin single variable domain, this may mean that said immunoglobulin single variable domain sequence has been incorporated into the sequence of the polypeptide of the present technology, but more usually this generally means that the polypeptide of the present technology contains within its sequence the sequence of the immunoglobulin single variable domains irrespective of how said polypeptide of the present technology has been generated or obtained. Also, when a nucleic acid or nucleotide sequence is said to comprise another nucleotide sequence, the first mentioned nucleic acid or nucleotide sequence is preferably such that, when it is expressed into an expression product (e.g. a polypeptide), the amino acid sequence encoded by the latter nucleotide sequence forms part of said expression product (in other words, that the latter nucleotide sequence is in the same reading frame as the first mentioned, larger nucleic acid or nucleotide sequence).

[0118] When an amino acid sequence or polypeptide is said to "essentially consist of" an immunoglobulin single variable domain, it is meant that said amino acid sequence or polypeptide either is exactly the same as the immunoglobulin single variable domain or corresponds to polypeptide or amino acid sequence which has a limited number of amino acid residues, such as 1-20 amino acid residues, for example 1-10 amino acid residues and preferably 1-6 amino acid residues, such as 1, 2, 3, 4, 5 or 6 amino acid residues, added at the amino terminal end, at the carboxy terminal end, or at both the amino terminal end and the carboxy terminal end of the immunoglobulin single variable domain. When "consist of" is used, it is meant that the amino acid sequence or polypeptide is exactly the same as the immunoglobulin single variable domain. It must be noted that as used herein, the singular forms "a", "an", and "the", include plural references unless the context clearly indicates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents and reference to "the method" includes reference to equivalent steps and methods known to those of ordinary skill in the art that could be modified or substituted for the methods described herein.

[0119] Unless otherwise indicated, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present technology described herein. Such equivalents are intended to be encompassed by the present technology.

[0120] The term "and / or" wherever used herein includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0121] The term "about" or "approximately" as used herein means within 20%, preferably within 15%, more preferably within 10%, and most preferably within 5% of a given value or range.

[0122] Amino acid residues will be indicated interchangeably herein according to the standard three-letter or one-letter amino acid code, as mentioned in Table A-l below.

[0123] Table A-l: Common amino acids

[0124]

[0125] When an amino acid residue is indicated as "X" or "Xaa", it means that the amino acid residue is unspecified, unless the context requires a more limited interpretation. For example, if the description provides an amino acid sequence of a CDR wherein one (or more) of the amino acid residue(s) is (are) indicated with "X", the description may further specify which amino acid residue(s) is (can be) present at that specific position of the CDR.

[0126] 1 Immunoglobulin single variable domains

[0127] The inventors, using HER2-binding ISVDs described in WO 2009 / 068625 A2, have created improved HER2- binding ISVDs with desirable binding properties. It was found that these ISVDs target epitopes that are different from trastuzumab and / or pertuzumab. Therefore, these ISVDs are very good candidates for being used in treatment of H ER2-positive cancers on their own and are also good candidates for being used in combination therapy with trastuzumab or pertuzumab.

[0128] In a first aspect the present technology relates to an immunoglobulin single variable domain (ISVD) which specifically binds to human and / or non-human primate HER2, wherein said ISVD essentially consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO:1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition wherein Xi is selected from I and S and X2is selected from V, R and Y; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition, wherein X3is selected from A and R and X4is selected from I, L and M; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XsIXeRVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition, wherein X5is selected from G and L, X6is selected from D and S and X7is selected from E and G; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein Xg is selected from D, E and V; or e. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIXi2SXi3GXi4PXi5 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is NG DXM (SEQ ID NO: 254), according to AbM definition, wherein

[0129] X3is selected from A and R;

[0130] X4is selected from I, L and M;

[0131] X9is selected from G and R;

[0132] X10 is selected from F and A;

[0133] Xu is selected from T and I;

[0134] Xi2is selected from R and G;

[0135] Xi3is selected from absence of an amino acid residue and S;

[0136] X14 is selected from A and T;

[0137] X15 is selected from V and A;

[0138] Xie is selected from I and Y; or f. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TXi7Xi2SXi3GXi4PXi5 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is NGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0139] X3is selected from A and R; X4is selected from I, L and M;

[0140] X9is selected from G and R;

[0141] Xio is selected from F and A;

[0142] Xu is selected from T and I;

[0143] X12 is selected from R and G;

[0144] X13 is selected from absence of an amino acid residue and S;

[0145] X14 is selected from A and T;

[0146] X15 is selected from V, Y and A;

[0147] Xi6 is selected from I and Y;

[0148] X17 is selected from I and S; and wherein the full amino acid sequence of the ISVD is different from any of SEQ ID NOs: 29-32.

[0149] In an embodiment a. the amino acid sequence of CDR1 is SEQ ID NO: 1, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 13-16, and the amino acid sequence of CDR3 is SEQ ID NO: 3, according to AbM definition; or b. the amino acid sequence of CDR1 is selected from SEQ ID NOs: 17-20, the amino acid sequence of CDR2 is SEQ ID NO: 5, and the amino acid sequence of CDR3 is SEQ ID NO: 6, according to AbM definition; or c. the amino acid sequence of CDR1 is SEQ ID NO: 7, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 21,23 and 24, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 22 and 25; according to AbM definition; or d. the amino acid sequence of CDR1 is SEQ ID NO: 10, the amino acid sequence of CDR2 is SEQ ID NO: 11, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 26-28; according to AbM definition.

[0150] In another embodiment a. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 13, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or b. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 14, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or c. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 15, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or d. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 16, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or e. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 17, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or f. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 18, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or g. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 19, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or h. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 20, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or i. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or

[0151] Y1 j. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or k. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or l. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or m. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or n. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or o. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 26; according to AbM definition; or p. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 27; according to AbM definition; or q. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 28; according to AbM definition. The term "immunoglobulin single variable domain" (ISVD), defines immunoglobulin molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain. This sets immunoglobulin single variable domains apart from "conventional" immunoglobulins (e.g. monoclonal antibodies) or their fragments (such as Fab, Fab', F(ab')2, scFv, di-scFv), wherein two immunoglobulin domains, in particular two variable domains, interact to form an antigen binding site. Typically, in conventional immunoglobulins, a heavy chain variable domain (VH) and a light chain variable domain (V ) interact to form an antigen binding site. In this case, the complementarity determining regions (CDRs) of both VH and V will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.

[0152] In view of the above definition, the antigen-binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-V pair of immunoglobulin domains, which jointly bind to an epitope of the respective antigen.

[0153] In contrast, immunoglobulin single variable domains are capable of specifically binding to an epitope of the antigen without pairing with an additional immunoglobulin variable domain. The binding site of an immunoglobulin single variable domain is formed by a single VH, a single VHH or single V domain.

[0154] As such, the single variable domain may be a light chain variable domain sequence (e.g., a V -sequence) or a suitable fragment thereof; or a heavy chain variable domain sequence (e.g., a VH-sequence or VHH sequence) or a suitable fragment thereof; as long as it is capable of forming a single antigen binding unit (i.e., a functional antigen binding unit that essentially consists of the single variable domain, such that the single antigen binding domain does not need to interact with another variable domain to form a functional antigen binding unit).

[0155] In yet another embodiment, the ISVD is a heavy-chain ISVD. In a further embodiment, the ISVD is selected from a VHH, a humanized VHH, a camelized VH, a domain antibody, a single domain antibody, and a dAb.

[0156] An immunoglobulin single variable domain (ISVD) can for example be a heavy chain ISVD, such as a VH, including a camelized VH or a VHH, including a humanized VHH. Preferably, it is a VHH, including a humanized VHH. Heavy chain ISVDs can be derived from a conventional four-chain antibody or from a heavy chain antibody.

[0157] For example, the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a single domain antibody), a "dAb" or dAb (or an amino acid sequence that is suitable for use as a dAb); other single variable domains, or any suitable fragment of any one thereof.

[0158] In particular, the immunoglobulin single variable domain may be a NANOBODY® immunoglobulin single variable domain (such as a VHH, including a humanized VHH, or camelized VH) or a suitable fragment thereof. NANOBODY® and NANOBODIES® are registered trademarks of Ablynx N.V.

[0159] "VHH domains", also known as VHHs, VHH antibody fragments, and VHH antibodies, have originally been described as the antigen binding immunoglobulin variable domain of "heavy chain antibodies" (i.e., of "antibodies devoid of light chains"; Hamers-Casterman et al. 1993, Nature 363: 446-448). The term "VHH domain" has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "VH domains") and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "V domains"). For a further description of VHH's, reference is made to the review article by Muyldermans 2001 (Reviews in Molecular Biotechnology 74: 277-302).

[0160] Typically, the generation of immunoglobulins involves the immunization of experimental animals, fusion of immunoglobulin producing cells to create hybridomas and screening for the desired specificities. Alternatively, immunoglobulins can be generated by screening of naive or synthetic libraries e.g. by phage display. The generation of immunoglobulin sequences, such as immunoglobulin single variable domains and VHH sequences, has been described extensively in various publications, among which WO 94 / 04678, Hamers- Casterman et al. 1993 and Muyldermans et al. 2001 can be exemplified. In these methods, camelids are immunized with the target antigen in order to induce an immune response against said target antigen. The repertoire of VHHs obtained from said immunization is further screened for VHHs that bind the target antigen.

[0161] In these instances, the generation of immunoglobulins, such as immunoglobulin single variable domains and VHHs, requires purified antigen for immunization and / or screening. Antigens can be purified from natural sources, or in the course of recombinant production. Immunization and / or screening for immunoglobulin sequences can be performed using peptide fragments of such antigens.

[0162] The present technology may use immunoglobulin sequences, such as immunoglobulin single variable domains, of different origin, comprising mouse, rat, rabbit, donkey, human and camelid immunoglobulin sequences. The technology also includes fully human, humanized, or chimeric sequences. For example, the technology comprises camelid immunoglobulin sequences and humanized camelid immunoglobulin sequences, or camelized domain antibodies, e.g. camelized dAb as described by Ward et al (see for example WO 94 / 04678 and Davies and Riechmann 1994 and 1996). Moreover, the technology also uses fused immunoglobulin sequences, e.g. forming a multivalent and / or multispecific construct (for multivalent and multispecific polypeptides containing one or more VHH domains and their preparation, reference is also made to Conrath et al. 2001, J. Biol. Chem. 276 (10): 7346-7350, as well as to for example WO 96 / 34103 and WO 99 / 23221), and immunoglobulin sequences comprising tags or other functional moieties, e.g. toxins, labels, radiochemicals, etc., which are derivable from the immunoglobulin sequences of the present technology.

[0163] A "humanized VHH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VHH domain, but that has been "humanized", i.e. by replacing one or more amino acid residues in the amino acid sequence of said naturally occurring VHH sequence (and in particular in the framework sequences) by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being (e.g. indicated above). This can be performed in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the prior art (e.g. WO 2008 / 020079). Again, it should be noted that such humanized VHHs can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VHH domain as a starting material.

[0164] A "camelized VH" comprises an amino acid sequence that corresponds to the amino acid sequence of a naturally occurring VH domain, but that has been "camelized", i.e. by replacing one or more amino acid residues in the amino acid sequence of a naturally occurring VH domain from a conventional 4-chain antibody by one or more of the amino acid residues that occur at the corresponding position(s) in a VHH domain of a heavy chain antibody. This can be performed in a manner known per se, which will be clear to the skilled person, for example based on the further description herein and the prior art (e.g. WO 2008 / 020079). Such "camelizing" substitutions are preferably inserted at amino acid positions that form and / or are present at the VH-V interface, and / or at the so-called Camelidae hallmark residues, as defined herein (see for example WO 94 / 04678 and Davies and Riechmann 1994 and 1996, supra). Preferably, the VH sequence that is used as a starting material or starting point for generating or designing the camelized VH is preferably a VH sequence from a mammal, more preferably the VH sequence of a human being, such as a VH3 sequence. However, it should be noted that such camelized VH can be obtained in any suitable manner known per se and thus are not strictly limited to polypeptides that have been obtained using a polypeptide that comprises a naturally occurring VH domain as a starting material.

[0165] A preferred structure of an immunoglobulin single variable domain sequence can be considered to be comprised of four framework regions ("FRs"), which are referred to in the art and herein as "Framework region 1" ("FR1"); as "Framework region 2" ("FR2"); as "Framework region 3" ("FR3"); and as "Framework region 4" ("FR4"), respectively; which framework regions are interrupted by three complementary determining regions ("CDRs"), which are referred to in the art and herein as "Complementarity Determining Region 1" ("CDR1"); as "Complementarity Determining Region 2" ("CDR2"); and as "Complementarity Determining Region 3" ("CDR3"), respectively.

[0166] As further described in paragraph q) on pages 58 and 59 of WO 08 / 020079, the amino acid residues of an immunoglobulin single variable domain can be numbered according to the general numbering for VH domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91), as applied to VHH domains from Camelids in the article of Riechmann and Muyldermans 1999 (J. Immunol. Methods 231: 25-38; see for example Figure 2 of this publication). It should be noted that - as is well known in the art for VH domains and for VHH domains - the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering). This means that, generally, the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence. The total number of amino acid residues in a VH domain and a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.

[0167] In the present application, unless indicated otherwise, CDR sequences were determined according to the AbM definition as described in Martin 2010 (In: Kontermann and Dubel (Eds.) 2010, Antibody Engineering, vol 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 comprises the amino acid residues at positions 1-25, CDR1 comprises the amino acid residues at positions 26-35, FR2 comprises the amino acids at positions 36-49, CDR2 comprises the amino acid residues at positions 50-58, FR3 comprises the amino acid residues at positions 59-94, CDR3 comprises the amino acid residues at positions 95-102, and FR4 comprises the amino acid residues at positions 103-113.

[0168] Determination of CDR regions may also be done according to different methods. One well-known method of determining CDR regions is according to Kabat (Martin 2010, In: Kontermann and Dubel (eds.), Antibody Engineering Vol. 2, Springer Verlag Heidelberg Berlin, Chapter 3, pp. 33-51). According to this method, FR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 1-30, CDR1 of an immunoglobulin single variable domain comprises the amino acid residues at positions 31-35, FR2 of an immunoglobulin single variable domain comprises the amino acids at positions 36-49, CDR2 of an immunoglobulin single variable domain comprises the amino acid residues at positions 50-65, FR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 66-94, CDR3 of an immunoglobulin single variable domain comprises the amino acid residues at positions 95-102, and FR4 of an immunoglobulin single variable domain comprises the amino acid residues at positions 103-113. In such an immunoglobulin sequence, the framework sequences may be any suitable framework sequences, and examples of suitable framework sequences will be clear to the skilled person, for example on the basis the standard handbooks and the further disclosure and prior art mentioned herein.

[0169] The framework sequences are preferably (a suitable combination of) immunoglobulin framework sequences or framework sequences that have been derived from immunoglobulin framework sequences (for example, by humanization or camelization). For example, the framework sequences may be framework sequences derived from a light chain variable domain (e.g. a V -sequence) and / orfrom a heavy chain variable domain (e.g. a VH-sequence or VHH sequence). In one particularly preferred aspect, the framework sequences are either framework sequences that have been derived from a VHH-sequence (in which said framework sequences may optionally have been partially or fully humanized) or are conventional VH sequences that have been camelized (as defined herein).

[0170] In particular, the framework sequences present in the ISVD sequence used in the technology may contain one or more of hallmark residues (as defined herein), such that the ISVD sequence is a VHH, including a humanized VHH or camelized VH. Some preferred, but non-limiting examples of (suitable combinations of) such framework sequences will become clear from the further disclosure herein.

[0171] Again, as generally described herein for the immunoglobulin sequences, it is also possible to use suitable fragments (or combinations of fragments) of any of the foregoing, such as fragments that contain one or more CDR sequences, suitably flanked by and / or linked via one or more framework sequences (for example, in the same order as these CDR's and framework sequences may occur in the full-sized immunoglobulin sequence from which the fragment has been derived).

[0172] However, it should be noted that the technology is not limited as to the origin of the ISVD sequence (or of the nucleotide sequence used to express it), nor as to the way that the ISVD sequence or nucleotide sequence is (or has been) generated or obtained. Thus, the ISVD sequences may be naturally occurring sequences (from any suitable species) or synthetic or semi-synthetic sequences. In a specific but nonlimiting aspect, the ISVD sequence is a naturally occurring sequence (from any suitable species) or a synthetic or semi-synthetic sequence, including but not limited to "humanized" (as defined herein) immunoglobulin sequences (such as partially or fully humanized mouse or rabbit immunoglobulin sequences, and in particular partially or fully humanized VHH sequences), "camelized" (as defined herein) immunoglobulin sequences, as well as immunoglobulin sequences that have been obtained by techniques such as affinity maturation (for example, starting from synthetic, random or naturally occurring immunoglobulin sequences), CDR grafting, veneering, combining fragments derived from different immunoglobulin sequences, PCR assembly using overlapping primers, and similar techniques for engineering immunoglobulin sequences well known to the skilled person; or any suitable combination of any of the foregoing.

[0173] Similarly, nucleotide sequences may be naturally occurring nucleotide sequences or synthetic or semisynthetic sequences, and may for example be sequences that are isolated by PCR from a suitable naturally occurring template (e.g. DNA or RNA isolated from a cell), nucleotide sequences that have been isolated from a library (and in particular, an expression library), nucleotide sequences that have been prepared by introducing mutations into a naturally occurring nucleotide sequence (using any suitable technique known per se, such as mismatch PCR), nucleotide sequence that have been prepared by PCR using overlapping primers, or nucleotide sequences that have been prepared using techniques for DNA synthesis known per se.

[0174] As described above, an ISVD may be an ISVD or a suitable fragment thereof. For a general description of ISVDs, reference is made to the further description below, as well as to the prior art cited herein. In this respect, it should however be noted that this description and the prior art mainly described ISVDs of the so-called "VH3 class" (i.e. ISVDs with a high degree of sequence homology to human germline sequences of the VH3 class such as DP-47, DP-51, or DP-29). It should however be noted that the technology in its broadest sense can generally use any type of ISVD, and for example also uses the ISVDs belonging to the so-called "VH4 class" (i.e. ISVDs with a high degree of sequence homology to human germline sequences of the VH4 class such as DP-78), as for example described in WO 2007 / 118670.

[0175] Generally, ISVDs (in particular VHH sequences, including (partially) humanized VHH sequences and camelized VH sequences) can be characterized by the presence of one or more "Hallmark residues" (as described herein) in one or more of the framework sequences (again as further described herein). Thus, generally, an ISVD can be defined as an immunoglobulin sequence with the (general) structure

[0176] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the Hallmark residues are as further defined herein.

[0177] In particular, an ISVD can be an immunoglobulin sequence with the (general) structure

[0178] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which the framework sequences are as further defined herein.

[0179] More in particular, an ISVD can be an immunoglobulin sequence with the (general) structure

[0180] FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4 in which FR1 to FR4 refer to framework regions 1 to 4, respectively, and in which CDR1 to CDR3 refer to the complementarity determining regions 1 to 3, respectively, and in which one or more of the amino acid residues at positions 11, 37, 44, 45, 47, 83, 84, 103, 104 and 108 according to the Kabat numbering are selected from the Hallmark residues mentioned in Table A-2 below.

[0181] Table A-2: Hallmark Residues in ISVDs

[0182] The technology inter alia uses ISVDs that can bind to H ER2. In the context of the present technology, "binding to" a certain target molecule has the usual meaning in the art as understood in the context of antibodies and their respective antigens.

[0183] The terms "specificity”, "binding specifically” or "specific binding” refer to the number of different target molecules, such as antigens, from the same organism to which a particular binding unit, such as an ISVD, can bind with sufficiently high affinity (see below). "Specificity”, "binding specifically” or "specific binding” are used interchangeably herein with "selectivity", "binding selectively” or "selective binding”. Binding units, such as ISVDs, preferably specifically bind to their designated targets. The specificity / selectivity of a binding unit can be determined based on affinity. The affinity denotes the strength or stability of a molecular interaction. The affinity is commonly given as by the KD, or dissociation constant, which is expressed in units of mol / liter (or M). The affinity can also be expressed as an association constant, KA, which equals 1 / KDand is expressed in units of (mol / liter)1(or M-1).

[0184] The affinity is a measure for the binding strength between a moiety and a binding site on the target molecule: the lower the value of the KD, the stronger the binding strength between a target molecule and a targeting moiety.

[0185] Typically, binding units used in the present technology (such as ISVDs) will bind to their targets with a dissociation constant (KD) of 10'5to 1012moles / liter or less, and preferably 10'7to 1012moles / liter or less and more preferably 10'8to 1012moles / liter (i.e. with an association constant (KA) of 105to 1012liter / moles or more, and preferably 107to 1012liter / moles or more and more preferably 108to 1012liter / moles).

[0186] Any KD value greater than 10'4mol / liter (or any KAvalue lower than 104liters / mol) is generally considered to indicate non-specific binding.

[0187] The KDfor biological interactions, such as the binding of immunoglobulin sequences to an antigen, which are considered specific are typically in the range of 10'5moles / liter (10000 nM or lOpM) to 1012moles / liter (0.001 nM or 1 pM) or less.

[0188] Accordingly, specific / selective binding may mean that - using the same measurement method, e.g. SPR - a binding unit (or polypeptide comprising the same) binds to HER2 with a KDvalue of 10'5to 1012moles / liter or less and binds to related targets with a KDvalue greater than 10'4moles / liter.

[0189] Specific binding to a certain target from a certain species does not exclude that the binding unit can also specifically bind to the analogous target from a different species. For example, specific binding to human HER2 does not exclude that the binding unit (or a polypeptide comprising the same) can also specifically bind to HER2 from cynomolgus monkeys.

[0190] When an ISVD is said to exhibit "improved cross-reactivity" for binding to human and non-human primate

[0191] HER2 compared to another ISVD, it means that for said ISVD the ratio of the binding activity (such as expressed in terms of KDor kOff) for human HER2 and for non-human primate HER2 is higher than that same ratio calculated for the other ISVD in the same assay.

[0192] Good cross-reactivity for binding to human and non-human primate HER2 allows for the assessment of toxicity of a polypeptide comprising said ISVD in preclinical studies conducted in non-human primates.

[0193] Specific binding of a binding unit to its designated target can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art; as well as the other techniques mentioned herein.

[0194] The dissociation constant may be the actual or apparent dissociation constant, as will be clear to the skilled person. Methods for determining the dissociation constant will be clear to the skilled person, and for example include the techniques mentioned below. In this respect, it will also be clear that it may not be possible to measure dissociation constants of more than 10'4moles / liter or 10'3moles / liter (e.g. of 10'2moles / liter). Optionally, as will also be clear to the skilled person, the (actual or apparent) dissociation constant may be calculated on the basis of the (actual or apparent) association constant (KA), by means of the relationship [KD= 1 / KA].

[0195] The affinity of a molecular interaction between two molecules can be measured via different techniques known per se, such as the well-known surface plasmon resonance (SPR) biosensor technique (see for example Ober et al. 2001, Intern. Immunology 13: 1551-1559). The term "surface plasmon resonance", as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, where one molecule is immobilized on the biosensor chip and the other molecule is passed over the immobilized molecule under flow conditions yielding kon, kOff measurements and hence KD(or KA) values. This can for example be performed using the well-known BIAcore® system (BIAcore International AB, a GE Healthcare company, Uppsala, Sweden and Piscataway, NJ). For further descriptions, see Jonsson et al. (1993, Ann. Biol. Clin. 51: 19-26), Jonsson et al. (1991, Biotechniques 11: 620-627), Johnsson et al. (1995, J. Mol. Recognit. 8: 125-131), and Johnnson et al. (1991, Anal. Biochem. 198: 268-277). Another well-known biosensor technique to determine affinities of biomolecular interactions is bio-layer interferometry (BLI) (see for example Abdiche et al. 2008, Anal. Biochem. 377: 209-217). The term "biolayer Interferometry" or "BLI", as used herein, refers to a label-free optical technique that analyzes the interference pattern of light reflected from two surfaces: an internal reference layer (reference beam) and a layer of immobilized protein on the biosensor tip (signal beam). A change in the number of molecules bound to the tip of the biosensor causes a shift in the interference pattern, reported as a wavelength shift (nm), the magnitude of which is a direct measure of the number of molecules bound to the biosensor tip surface. Since the interactions can be measured in real-time, association and dissociation rates and affinities can be determined. BLI can for example be performed using the well-known Octet® Systems (ForteBio, a division of Pall Life Sciences, Menlo Park, USA).

[0196] Alternatively, affinities can be measured in Kinetic Exclusion Assay (KinExA) (see for example Drake et al. 2004, Anal. Biochem., 328: 35-43), using the KinExA® platform (Sapidyne Instruments Inc, Boise, USA). The term "KinExA", as used herein, refers to a solution-based method to measure true equilibrium binding affinity and kinetics of unmodified molecules. Equilibrated solutions of an antibody / antigen complex are passed over a column with beads precoated with antigen (or antibody), allowing the free antibody (or antigen) to bind to the coated molecule. Detection of the antibody (or antigen) thus captured is accomplished with a fluorescently labeled protein binding the antibody (or antigen).

[0197] The GYROLAB® immunoassay system provides a platform for automated bioanalysis and rapid sample turnaround (Fraley et al. 2013, Bioanalysis 5: 1765-74).

[0198] Thus, in an embodiment, the ISVD has an affinity for human and / or non-human primate HER2 of between 1-600 nM, preferably between 1-500 nM, or between 1-400 nM, or between 1-300 nM, more preferably between 1-100 nM, most preferably between 1-10 nM.

[0199] In order to have a T cell engager effectively kill tumor cells but not kill other normal human cells with, e.g., the same HER2 expression levels, it could be desirable that the ISVD binds to the tumor target, specifically HER2, with different (such as lower) affinity. These different (such as lower) affinity HER2-binding ISVDs can then be linked to provide avidity binding bivalent ISVDs. Avidity binding by such HER2-binding ISVDs linked together may be facilitated in enriched HER2 areas which are present on a tumor cell, while in nonenriched areas on normal human cells no binding will be observed by these different affinity binding ISVDs. Such avidity binding in the enriched HER2 areas on a tumor cell will lead to better T cell dependent killing in the tumor, while no T cell dependent killing will be observed in non-enriched normal cell areas.

[0200] In some embodiments, the present technology provides ISVDs with different affinities (Table 10), including a high (< 10 nM), a medium (about 15-about 110 nM) and a low (>110 nM) affinity variant for each of the selected ISVDs, as determined e.g. by SPR. ISVDs with a high potency include A032800154 (SEQ ID NO: 33), A032801483 (SEQ ID NO:36), A032801467 (SEQ ID NO: 40). ISVDs with a medium potency include A032801524 (SEQ ID NO: 34), A032801484 (SEQ ID NO: 37), A032801441 (SEQ ID NO: 39), A032801497 (SEQ ID NO: 41). ISVDs with lower potency include A032801529 (SEQ ID NO: 35), A032801579 (SEQ ID NO: 38), A032801499 (SEQ ID NO: 42).

[0201] In a further embodiment, the present technology provides ISVDs that have been sequence optimized. These sequence optimizations may include:

[0202] 1) Substitutions in parental wild type ISVD sequences to yield ISVD sequences that are more identical to the human VH3-JH germline consensus sequences, a process called humanization. To this end, specific amino acids, with the exception of the so-called hallmark residues, in the FRs that differ between the ISVD and the human VH3-JH germline consensus are altered to the human counterpart in such a way that the protein structure, activity and stability are kept intact.

[0203] 2) Substitutions towards the llama germline to increase the stability of the ISVD, which is defined as camelisation. To this end, the parental wild type ISVD amino acid sequence is aligned to the llama IGHV germline amino acid sequence of the ISVD (identified as the top hit from a BlastP analysis of the ISVD against the llama IGHV germlines).

[0204] 3) Substitutions that improve long-term stability or properties under storage, substitutions that increase expression levels in a desired host cell or host organism, and / or substitutions that remove or reduce (undesired) post-translational modification(s) (such as glycosylation or phosphorylation), again depending on the desired host cell or host organism.

[0205] Examples of sequence optimized ISVDs according to the present technology are A032800154 (SEQ ID NO: 33), A032801524 (SEQ ID NO: 34), A032801529 (SEQ ID NO: 35), A032801483 (SEQ ID NO: 36), A032801484 (SEQ ID NO: 37), A032801579 (SEQ ID NO: 38), A032801441 (SEQ ID NO: 39), A032801467 (SEQ ID NO: 40), A032801497 (SEQ ID NO: 41), A032801499(SEQ ID NO: 42), A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801663 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), and A032801593(SEQ ID NO: 53).

[0206] In an embodiment, the present technology provides ISVDs that have, besides sequence optimization, further been subjected to T cell epitope de-risking in order to minimize the risk of anti-drug antibodies (ADAs) being triggered. T cell epitope de-risked ISVDs include A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801663 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), and A032801593(SEQ ID NO: 53).

[0207] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0208] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0209] The percentage of "sequence identity” between a first amino acid sequence and a second amino acid sequence may be calculated by dividing [the number of amino acid residues in the first amino acid sequence that are identical to the amino acid residues at the corresponding positions in the second amino acid sequence] by [the total number of amino acid residues in the first amino acid sequence] and multiplying by [100%], in which each deletion, insertion, substitution or addition of an amino acid residue in the second amino acid sequence - compared to the first amino acid sequence - is considered as a difference at a single amino acid residue (i.e. at a single position).

[0210] Usually, for the purpose of determining the percentage of "sequence identity" between two amino acid sequences in accordance with the calculation method outlined hereinabove, the amino acid sequence with the greatest number of amino acid residues will be taken as the "first" amino acid sequence, and the other amino acid sequence will be taken as the "second" amino acid sequence. An "amino acid difference" as used herein refers to a deletion, insertion, or substitution of a single amino acid residue vis-a-vis a reference sequence, and preferably is a substitution.

[0211] In one embodiment, amino acid substitutions are conservative substitutions. Such conservative substitutions preferably are substitutions in which one amino acid within the following groups (a) - (e) is substituted by another amino acid residue within the same group: (a) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (b) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and Gin; (c) polar, positively charged residues: His, Arg and Lys; (d) large aliphatic, nonpolar residues: Met, Leu, He, Vai and Cys; and (e) aromatic residues: Phe, Tyr and Trp.

[0212] In another embodiment of the present technology, the conservative substitutions are as follows: Ala into Gly or into Ser; Arg into Lys; Asn into Gin or into His; Asp into Glu; Cys into Ser; Gin into Asn; Glu into Asp; Gly into Ala or into Pro; His into Asn or into Gin; He into Leu or into Vai; Leu into lie or into Vai; Lys into Arg, into Gin or into Glu; Met into Leu, into Tyr or into lie; Phe into Met, into Leu or into Tyr; Ser into Thr; Thr into Ser; Trp into Tyr; Tyr into Trp; and / or Phe into Vai, into lie or into Leu.

[0213] A list of generated ISVDs can be found in Table A-3. Additionally, combinations of CDR sequences, according to the AbM definition, of the generated ISVDs can be found in Table A-4.

[0214] Table A-3: Newly generated HER2-binding ISVDs

[0215] Table A-4: CDRs of HER2-binding ISVDs ("ID" refers to the given SEQ ID NO)

[0216] In an embodiment, the ISVD has a CDR1, CDR2 and CDRS sequence selected from the CDR1, CDR2 and CDRS sequences presented in Table A-4.

[0217] In another embodiment, the ISVD has a CDR1, CDR2 and CDRS sequence selected from the combination of CDR sequences presented in the same row in Table A-4.

[0218] The framework sequences of the generated ISVDs can be found in Table A-5. Each line corresponds with the CDRs in the same line as in Table A-4 to make up the full sequence of the ISVD. Table A-5: Framework sequences of HER2-binding ISVDs ("ID" refers to the given SEQ ID NO)

[0219] In an embodiment, the ISVD comprises (in addition to the CDRs as defined above) one or more, or all, of the framework regions as indicated in Table A-5.

[0220] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0221] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032800154 (SEQ ID NO: 33) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0222] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801524 (SEQ ID NO: 34) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0223] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801529 (SEQ ID NO: 35) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded. In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801483 (SEQ ID NO: 36) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0224] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801484 (SEQ ID NO: 37) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0225] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801579 (SEQ ID NO: 38) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0226] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801441 (SEQ ID NO: 39) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0227] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801467 (SEQ ID NO: 40) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0228] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801497 (SEQ ID NO: 41) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0229] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801499 (SEQ ID NO: 42) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0230] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801618 (SEQ ID NO: 43) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0231] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801621 (SEQ ID NO: 44) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0232] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801623 (SEQ ID NO: 45) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0233] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801663 (SEQ ID NO: 46) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0234] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801668 (SEQ ID NO: 47) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0235] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801613 (SEQ ID NO: 48) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded. In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801689 (SEQ ID NO: 49) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0236] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801690 (SEQ ID NO: 50) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0237] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801588 (SEQ ID NO: 51) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0238] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801589 (SEQ ID NO: 52) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0239] In another embodiment, the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with A032801593 (SEQ ID NO: 53) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0240] In yet a further embodiment, the ISVD comprises or consists of a sequence selected from SEQ ID NOs: 33- 53.

[0241] In one embodiment, the ISVD comprises or consists of the full amino acid sequence of A032800154 (SEQ ID NO: 33), A032801524 (SEQ ID NO: 34), A032801529 (SEQ ID NO: 35), A032801483 (SEQ ID NO: 36), A032801484 (SEQ ID NO: 37), A032801579 (SEQ ID NO: 38), A032801441 (SEQ ID NO: 39), A032801467 (SEQ ID NO: 40), A032801497 (SEQ ID NO: 41), A032801499 (SEQ ID NO: 42), A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801663 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), or A032801593 (SEQ ID NO: 53) (Table A-3).

[0242] As will be clear from the further description above and herein, the ISVDs of the present technology can be used as "building blocks" to form polypeptides of the present technology, e.g., by suitably combining them with other groups, residues, moieties or binding units, in order to form compounds or constructs as described herein (such as, without limitations, the mono- / bi- / tri- / tetra- / multivalent and bi- / tri- / tetra- / multispecific polypeptides of the present technology described further herein), which combine within one molecule one or more desired properties or biological functions. As such, the present technology also relates to polypeptides that comprise one or more ISVDs of the present technology.

[0243] 2 Polypeptides

[0244] The inventors found that using the HER2-binding ISVDs in a polypeptide format had several advantages. Using ISVDs with different (such as lower) affinity for HER2, led to polypeptides that could effectively target tumor cells only expressing (comparatively) low levels of HER2, while normal human cells, also expressing HER2, were not targeted. Additionally, by using strategies such as T cell epitope de-risking and sequence optimization, the polypeptides were less likely to trigger anti-drug antibodies (ADAs) or pre-existing antibody binding against the polypeptide.

[0245] Thus, an aspect of the present technology relates to a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR sequences selected from the following: a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO: 1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XsIXeRVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein

[0246] Xi is selected from I and S;

[0247] X2is selected from V, R and Y;

[0248] X3is selected from A and R;

[0249] X4is selected from I, L and M;

[0250] X5is selected from G and L;

[0251] Xe is selected from D and S;

[0252] X7is selected from E and G;

[0253] X8is selected from D, E and V; or e. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIXi2SXi3GXi4PXi5 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is NG DXM (SEQ ID NO: 254), according to AbM definition, wherein

[0254] X3is selected from A and R;

[0255] X4is selected from I, L and M;

[0256] X9is selected from G and R;

[0257] X10 is selected from F and A;

[0258] Xu is selected from T and I;

[0259] X12 is selected from R and G;

[0260] Xi3is selected from absence of an amino acid residue and S;

[0261] X14 is selected from A and T;

[0262] X15 is selected from V, Y and A;

[0263] Xie is selected from I and Y; or f. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TX17X12SX13GX14PX15 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is NGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0264] X3is selected from A and R;

[0265] X4is selected from I, L and M;

[0266] X9is selected from G and R;

[0267] X10 is selected from F and A;

[0268] Xu is selected from T and I;

[0269] X12 is selected from R and G;

[0270] Xi3is selected from absence of an amino acid residue or S;

[0271] X14 is selected from A and T;

[0272] X15 is selected from V, Y and A;

[0273] Xi6 is selected from I and Y;

[0274] X17 is selected from I and S; and wherein the second ISVD has a sequence that may be the same or different from the first ISVD.

[0275] In some embodiments, the present technology relates to a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR sequences selected from the following: a. the amino acid sequence of CDR1 is SEQ ID NO: 1, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 13-16, and the amino acid sequence of CDR3 is SEQ ID NO: 3, according to AbM definition; or b. the amino acid sequence of CDR1 is selected from SEQ ID NOs: 17-20, the amino acid sequence of CDR2 is SEQ ID NO: 5, and the amino acid sequence of CDR3 is SEQ ID NO: 6, according to AbM definition; or c. the amino acid sequence of CDR1 is SEQ ID NO: 7, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 21, 23 and 24, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 22 and 25; according to AbM definition; or d. the amino acid sequence of CDR1 is SEQ ID NO: 10, the amino acid sequence of CDR2 is SEQ ID

[0276] NO: 11, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 26-28; according to

[0277] AbM definition.

[0278] In another embodiment, the present technology relates to a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR sequences selected from the following: a. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 13, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or b. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 14, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or c. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 15, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or d. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 16, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or e. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 17, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or f. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 18, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or g. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 19, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or h. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 20, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or i. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or j. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or k. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or l. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or m. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or n. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or o. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 26; according to AbM definition; or p. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 27; according to AbM definition; or q. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 28; according to AbM definition.

[0279] In some embodiments, the present technology relates to a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR1, CDR2 and CDR3 sequence selected from the CDR1, CDR2 and CDR3 sequences presented in Table A-4.

[0280] In another embodiment, the present technology relates to a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR1, CDR2 and CDR3 sequence selected from the combination of CDR sequences presented in the same row in Table A-4.

[0281] Specific examples of such ISVDs comprised in the polypeptide of the present technology have one or more, or all, framework regions as indicated in Table A-5 (in addition to the CDRs as defined above). Specific examples of such ISVDs comprised in the polypeptide of the present technology have one or more, or all, framework regions as indicated in the same row in Table A-5 (in addition to the CDRs as defined above) for A032800154 (SEQ ID NO: 33), A032801524 (SEQ ID NO: 34), A032801529 (SEQ ID NO: 35), A032801483 (SEQ ID NO: 36), A032801484 (SEQ ID NO: 37), A032801579 (SEQ ID NO: 38), A032801441 (SEQ ID NO: 39), A032801467 (SEQ ID NO: 40), A032801497 (SEQ ID NO: 41), A032801499 (SEQ ID NO: 42), A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801663 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), or A032801593 (SEQ ID NO: 53).

[0282] Specific examples of such ISVDs comprised in the polypeptide of the present technology comprise or essentially consist of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0283] Specific examples of such ISVDs comprised in the polypeptide of the present technology comprise or consist of a sequence selected from SEQ ID NOs: 29-53.

[0284] In one embodiment, the ISVD comprised in the polypeptide of the present technology consists of the full amino acid sequence of 27A05 (SEQ ID NO: 29), 29E09 (SEQ ID NO: 30), 47D05 (SEQ ID NO: 31), 27E07 (SEQ ID NO: 32), A032800154 (SEQ ID NO: 33), A032801524 (SEQ ID NO: 34), A032801529 (SEQ ID NO: 35), A032801483 (SEQ ID NO: 36), A032801484 (SEQ ID NO: 37), A032801579 (SEQ ID NO: 38), A032801441 (SEQ ID NO: 39), A032801467 (SEQ ID NO: 40), A032801497 (SEQ ID NO: 41), A032801499 (SEQ ID NO: 42), A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801663 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), or A032801593 (SEQ ID NO: 53) (Table A-3).

[0285] In an embodiment of the present technology, the polypeptide is monospecific, wherein the polypeptide comprises or essentially consists of at least two ISVDs capable of binding specifically human and / or nonhuman primate HER2, and wherein the HER2 is preferably human. For example, the polypeptide may be monospecific-bivalent, such as a polypeptide comprising or essentially consisting of two ISVDs, wherein both ISVDs bind to human and / or non-human primate HER2, and wherein the HER2 is preferably human.

[0286] Such a polypeptide may at the same time be biparatopic, for example if two ISVDs bind two different epitopes of the target antigen, such as two epitopes on human and / or non-human primate HER2, preferably human HER2.

[0287] The term "biparatopic" refers to binding to two different parts (e.g., epitopes) of the same target molecule.

[0288] Thus, in an embodiment, the polypeptide comprises or essentially consists of at least 2 ISVDs, wherein both ISVDs bind to human and / or non-human primate HER2, wherein the two ISVDs target different epitopes on human and / or non-human primate HER2, and wherein the HER2 is preferably human.

[0289] In an embodiment, the first and second ISVD have sequences that are selected from sequences that have a degree of sequence identity with one of the sequences of SEQ ID NOs: 29-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0290] In an embodiment, the first and / or the second ISVD have sequences that are selected from sequences that have a degree of sequence identity with one of the sequences of SEQ ID NOs: 29-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0291] In another embodiment, the first ISVD has a sequence selected from SEQ ID NOs: 29-53.

[0292] In a further embodiment, the second ISVD has a sequence selected from SEQ ID NOs: 29-53.

[0293] In another embodiment, the polypeptide is multispecific, such as at least bispecific, but can also be e.g., trispecific, tetraspecific, pentaspecific, etc. Moreover, the polypeptide is multivalent, such as at least bivalent, but can also be e.g., trivalent, tetravalent, pentavalent, hexavalent, etc. The terms “bispecific", “trispecific", “tetraspecific", “pentaspecific", etc., all fall under the term "multispecific” and refer to binding to two, three, four, five, etc., different target molecules, respectively. The terms "bivalent”, "trivalent”, "tetravalent”, "pentavalent”, "hexavalent”, etc. all fall under the term "multivalent” and indicate the presence of two, three, four, five, six, etc., binding units / building blocks, respectively, such as ISVDs.

[0294] Thus, in an embodiment, the polypeptide is bispecific and at least bivalent, wherein the polypeptide comprises or essentially consists of at least two ISVDs, wherein the first ISVD is capable of binding specifically to human and / or non-human primate HER2 and the second ISVD is capable of binding specifically to the constant domain of human and / or non-human primate TCR, and wherein the HER2 and TCR are preferably human.

[0295] As another example, the polypeptide could be bispecific-trivalent, wherein the polypeptide comprises or essentially consists of three ISVDs, wherein the first and second ISVD are capable of binding specifically to human and / or non-human primate HER2 and the third ISVD is capable of binding specifically to the constant domain of human and / or non-human primate TCR, and wherein the HER2 and TCR are preferably human.

[0296] Consequently, in yet another embodiment, the polypeptide further comprises a third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell.

[0297] The inventors found that by using a T cell engaging (TCE) ISVD, the polypeptide can trigger effective HER2- positive tumor cell killing through T cell dependent cytotoxicity (TDCC). This was particularly effective using a TCE ISVD that was targeting the T cell receptor (TCR).

[0298] Binding to TCR can be achieved, for example, by binding to the TCRalpha subunit and / or the TCRbeta subunit. An example is mammalian TCR. While human TCR is preferred, the versions from other species are also amenable to the present technology, for example TCR from mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys (also referred to herein as "cyno”), or camelids, such as llama or alpaca. The sequences of the TCR-a / 0 constant domains of human and cyno origin are provided in Table A-7 (SEQ ID NO: 118 and 123 for the constant domain of TCR a from human and cyno origin, respectively; SEQ ID NO: 119 and 124 for the constant domain of TCR 0 from human and cyno origin, respectively). The origin of each of these sequences, as expressed by a UniProt or Genbank files identifier, is listed for each of the aforementioned sequences in Table A-7. In house sequencing confirmed that the amino acid sequences originally derived from rhesus origin, were identical to those from cyno origin.

[0299] In one embodiment, the TCE ISVD targeting the TCR (e.g., the second or third ISVD) specifically binds to the constant domain of a human T cell receptor a (TCR-a) (SEQ ID NO: 118) and / or the constant domain of the human T cell receptor 0 (TCR-0) (SEQ ID NO: 119), or polymorphic variants or isoforms thereof.

[0300] In one embodiment, the TCE ISVD targeting the TCR (e.g., the second or third ISVD) specifically binds to the constant domain of a non-human primate TCR. In one embodiment, the non-human primate TCR is a macaque or rhesus TCR. In one embodiment, the macaque or rhesus TCR comprises the constant domain of a TCR-a of SEQ ID NO: 120 and / or of a TCR-0 of SEQ ID NO: 121, or polymorphic variants or isoforms thereof.

[0301] Isoforms are alternative protein sequences that can be generated from the same gene by a single or by the combination of biological events such as alternative promoter usage, alternative splicing, alternative initiation and ribosomal frameshifting, all as known in the art.

[0302] Table A-7: Amino acid sequences related to TCR ("ID" refers to the given SEQ ID NO as used herein) Examples of TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology can be found in WO 2016 / 180969, WO 2022 / 129637, and WO 2023 / 242247.

[0303] In an embodiment, the TCE ISVD has a CDR1 with amino acid sequence XIX2VHKINFX3G (SEQ ID NO: 214), a CDR2 with amino acid sequence HISIGDQTD (SEQ ID NO: 215) and a CDR3 with amino acid sequence X4SRIX5PYDY (SEQ ID NO: 216), according to Abm definition, wherein

[0304] - Xi is selected from G, W, E and D;

[0305] - X2is selected from D, Y G and W;

[0306] - X3is selected from L and Y;

[0307] - X4is selected from F and L; and

[0308] - X5is selected from W, Y, T, S and Q.

[0309] In some embodiments, the TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 219.

[0310] In some embodiments, the TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY).

[0311] In some embodiments, the TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM) definition that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIYPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 259.

[0312] In some embodiments, the TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIWPYDY).

[0313] Specific examples of TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology have one or more, or all, framework regions as indicated in Table A-8 (in addition to the CDRs as defined above). Specific examples of TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology have one or more, or all, framework regions as indicated in Table A-8 (in addition to the CDRs as defined above) for TCE01 (SEQ ID NO: 55 or 265), TCE03a (SEQ ID NO: 56 or 268).

[0314] Table A-8: Sequences for CDRs and frameworks ("ID" refers to the given SEQ ID NO; "Seq" refers to the sequence), for the TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology, using the AbM definition Further examples of TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology are the following TCE ISVDs as depicted in Table A-8a.

[0315] Table A-8a: Sequences for the TCE ISVDs that may be used as the second and / or third ISVD in the polypeptide of the present technology

[0316] In an embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with SEQ ID NO: 55 (TCE01), SEQ ID NO: 265 (TCE01 (EID)), SEQ ID NO: 268 (TCE03a) or SEQ ID NO: 56 (TCE03a(ElD)) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0317] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE01 (SEQ ID NO: 55) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0318] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE03a(ElD) (SEQ ID NO: 56) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0319] In an embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with SEQ ID NO: 55 (TCE01), SEQ ID NO: 265 (TCE01 (EID)), SEQ ID NO: 268 (TCE03a) or SEQ ID NO: 56 (TCE03a(ElD)) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%. In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE01 (SEQ ID NO: 55) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0320] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE03a(ElD) (SEQ ID NO: 56) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0321] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55, SEQ ID NO: 265 (TCE01 (EID)), SEQ ID NO: 268 (TCE03a) and SEQ ID NO: 56.

[0322] In an embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NO: 261 to SEQ ID NO.: 268 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0323] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE688 (SEQ ID NO: 261) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0324] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE688 (EID) (SEQ ID NO: 262) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded. In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE 56G05 (SEQ ID NO: 263) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0325] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE 56G05 (EID) (SEQ ID NO: 264) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0326] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE01 (EID) (SEQ ID NO: 265) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0327] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE02 (SEQ ID NO: 266) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0328] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE02 (EID) (SEQ ID NO: 267) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0329] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE03a (SEQ ID NO: 268) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0330] In an embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity any one of SEQ ID NO: 261 to SEQ ID NO.: 268 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0331] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE688 (SEQ ID NO: 261) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0332] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE688 (EID) (SEQ ID NO: 262) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0333] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE 56G05 (SEQ ID NO: 263) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0334] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE 56G05 (EID) (SEQ ID NO: 264) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0335] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE01 (EID) (SEQ ID NO: 265) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%. In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE02 (SEQ ID NO: 266) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0336] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE02 (EID) (SEQ ID NO: 267) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0337] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with TCE03a (SEQ ID NO: 268) of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0338] In another embodiment, the TCE ISVDs that may be used as the second and / or third ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NO: 261 to SEQ ID NO.: 268.

[0339] The inventors found that having a TCE ISVD at the N-terminal end of the polypeptide resulted in the best T cell activation. Thus, in a further embodiment, the second and / or third ISVD is at the N-terminal end of the polypeptide.

[0340] The technology envisages various combination of first, second and / or third ISVD as long as the resulting polypeptide provides for effective tumor cell killing, even killing of low HER2 expressing tumor cells.

[0341] In an embodiment, the polypeptide comprises: a. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 31, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or b. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or c. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or d. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or e. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or f. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 34, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or g. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or h. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 41 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or i. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or j. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 50, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or k. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or l. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or m. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or n. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or o. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or p. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 40, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or q. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or r. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or s. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or t. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 51, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or u. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or v. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 45, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or w. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56.

[0342] The combinations of these specific ISVDs were found to confer the desirable properties, e.g., binding to HER2 and activating a T cell response, to the polypeptides of the present technology.

[0343] In particular, the combination of a first ISVD with the sequence of SEQ ID NO: 48, a second ISVD with the sequence of SEQ ID NO: 53 or SEQ ID NO: 47, and a third ISVD with the sequence of SEQ ID NO: 56, were found to be especially effective in tumor cell killing, even in tumor cells expressing low levels of HER2.

[0344] Therefore, in an embodiment, the polypeptide comprises a. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or b. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56.

[0345] SEQ ID NO.: 56 corresponds to SEQ ID NO.: 268 with the only difference that the first amino acid is E in SEQ ID NO.: 268 and D in SEQ ID NO.: 56. SEQ ID NO.: 55 corresponds to SEQ ID NO.: 265 with the only difference that the first amino acid is E in SEQ ID NO.: 55 and D in SEQ ID NO.: 265.

[0346] Therefore, in another embodiment the polypeptide comprises: a. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 31, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or b. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or c. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or d. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or e. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or f. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 34, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or g. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or h. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 41 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or i. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or j. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 50, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or k. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or l. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or m. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or n. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or o. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or p. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 40, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or q. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or r. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or s. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or t. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 51, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or u. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or v. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 45, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or w. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268.

[0347] In another embodiment, the polypeptide comprises a. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or b. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268.

[0348] In one embodiment, the polypeptide may further comprise one or more other groups, residues, moieties or binding units, optionally linked via one or more peptidic linkers, in which said one or more other groups, residues, moieties or binding units provide the polypeptide with increased (in vivo) half-life, compared to the corresponding polypeptide without said one or more other groups, residues, moieties or binding units.

[0349] In vivo half-life extension means, for example, that the polypeptide has an increased half-life in a mammal, such as a human subject, after administration. Half-life can be expressed for example as tl / 2beta. This will be further elaborated on in the section "(In vivo) half-life extension".

[0350] More specifically, said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life can be chosen from the group consisting of binding units that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, said one or more other groups, residues, moieties or binding units that provide the polypeptide with increased half-life is a binding unit that can bind to serum albumin, such as human serum albumin, or a serum immunoglobulin, such as IgG. In one embodiment, the binding unit is an ISVD.

[0351] For example, WO 2004 / 041865 and WO 2006 / 122787 describes ISVDs binding to serum albumin (and in particular against human serum albumin) that can be linked to other proteins (such as one or more other ISVDs binding to a desired target) in order to increase the half-life of said protein. These ISVDs include the ISVDs called Alb-1 (SEQ ID NO: 52 in WO 2006 / 122787) and humanized variants thereof, such as Alb-8 (SEQ ID NO: 62 in WO 2006 / 122787). Again, these can be used to extend the half-life of therapeutic proteins and polypeptide and other therapeutic entities or moieties. Moreover, WO 2012 / 175400 describes a further improved version of Alb-1, called Alb-23.

[0352] In an embodiment, the polypeptide comprises a serum albumin binding moiety selected from Alb-1, Alb- 3, Alb-4, Alb-5, Alb-6, Alb-7, Alb-8, Alb-9, Alb-10 and Alb-23., In one embodiment, the serum albumin binding moiety is Alb-8 or Alb-23 or its variants, as shown in pages 7-9 of WO 2012 / 175400.

[0353] In a further embodiment, the serum albumin binding moiety is selected from the albumin binders described in WO 2012 / 175741, WO 2015 / 173325, WO 2017 / 080850, WO 2017 / 085172, WO 2018 / 104444, WO 2018 / 134235, and WO 2018 / 134234. Some serum albumin binders are also shown in Table A-9.

[0354] In an embodiment, a further component of the polypeptide is an ISVD that specifically binds to human serum albumin and comprises i. a CDR1 that has the sequence GFTFRSFGMS (SEQ ID NO: 220) or an amino acid sequence with 2 or 1 amino acid difference with GFTFRSFGMS (according to AbM definition); ii. a CDR2 that has the sequence SISGSGSDTL (SEQ ID NO: 221) or an amino acid sequence with 2 or 1 amino acid difference with SISGSGSDTL (according to AbM definition); and ill. a CDR3 that has the sequence GGSLSR (SEQ ID NO: 222) or an amino acid sequence with 2 or 1 amino acid difference with GGSLSR (according to AbM definition).

[0355] In one embodiment, the ISVD that specifically binds to human serum albumin comprises a CDR1 that is the amino acid sequence of SEQ ID NO: 220, a CDR2 that is the amino acid sequence of SEQ ID NO: 221 and a CDR3 that is the amino acid sequence of SEQ ID NO: 222.

[0356] Also in another embodiment, the amino acid sequence of an ISVD binding to human serum albumin may have a sequence identity of more than 90%, such as more than 95% or more than 99%, with SEQ ID NO: 114, wherein the CDRs are as defined above. In one embodiment, the ISVD binding to human serum albumin comprises or consists of the amino acid sequence of SEQ ID NO: 114. When such an ISVD binding to human serum albumin has 2 or 1 amino acid difference in at least one CDR relative to a corresponding reference CDR sequence (as defined above), the ISVD has at least half of the binding affinity, or at least the same binding affinity, to human serum albumin compared to construct ALB23002 (SEQ ID NO: 114), wherein the binding affinity is measured using the same method, such as SPR.

[0357] In another embodiment, the polypeptide comprises a serum albumin binding moiety selected from Table A-9, such as e.g. a sequence selected from SEQ ID NO: 100 or 114, such as SEQ ID NO: 114.

[0358] Table A-9: Serum albumin binding ISVD sequences ("ID" refers to the SEQ ID NO as used herein)

[0359] In an embodiment, the polypeptide may be trispecific-trivalent, such a polypeptide comprising or consisting of three ISVDs, wherein one ISVD specifically binds to the constant domain of a human and / or a non-human primate TCR on a T cell, one ISVD specifically binds to a human or non-human primate HER2, and one ISVD provides in vivo half-life extension, and wherein the TCR and HER2 are preferably human.

[0360] In an embodiment, the polypeptide may be trispecific-tetravalent, such a polypeptide comprising or consisting of four ISVDs, wherein one ISVD specifically binds to the constant domain of a human and / or a non-human primate TCR on a T cell, two ISVDs specifically bind to a human or non-human primate HER2, and one ISVD provides in vivo half-life extension, and wherein the TCR and HER2 are preferably human.

[0361] In an embodiment, the third or fourth ISVD that provides in vivo half-life extension specifically binds to human serum albumin comprises i. a CDR1 that has the sequence GFTFRSFGMS (SEQ ID NO: 220) or an amino acid sequence with 2 or 1 amino acid difference with GFTFRSFGMS (according to AbM definition); ii. a CDR2 that has the sequence SISGSGSDTL (SEQ ID NO: 221) or an amino acid sequence with 2 or 1 amino acid difference with SISGSGSDTL (according to AbM definition); and ill. a CDR3 that has the sequence GGSLSR (SEQ ID NO: 222) or an amino acid sequence with 2 or 1 amino acid difference with GGSLSR (according to AbM definition).

[0362] In an embodiment, the third or fourth ISVD that provides / / ? vivo half-life extension comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 100-117 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded, preferably a degree of sequence identity with SEQ ID NO: 114 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0363] In an embodiment, the third or fourth ISVD that provides / / ? vivo half-life extension comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 100-117 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded, preferably a degree of sequence identity with SEQ ID NO: 114 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%%.

[0364] In a further embodiment, the third or fourth ISVD that provides in vivo half-life extension comprises or essentially consists of SEQ ID NO: 114.

[0365] In another embodiment, the third or fourth ISVD that provides in vivo half-life extension comprises or essentially consists of a sequence that has a degree of sequence identity with SEQ ID NO: 102 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0366] In yet a further embodiment, the third or fourth ISVD that provides in vivo half-life extension comprises or essentially consists of SEQ ID NO: 102. When such an ISVD binding to human serum albumin is at a C-terminal position it can exhibit a C-terminal alanine (A) or glycine (G) extension (preferably A). In some embodiments, the ISVD binding to human serum albumin is at another position than the C-terminal position (i.e. is not the C-terminal ISVD of the polypeptide).

[0367] In another embodiment, the polypeptide has an affinity for human and / or non-human primate HER2 of between 1-600 nM, preferably between 1-500 nM, more preferably between 1-100 nM, most preferably between 1-10 nM.

[0368] In an embodiment, the sequence of the polypeptide comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 62-67 or 76-85 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0369] In an embodiment, the sequence of the polypeptide comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 62-67 or 76-85 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0370] In a further embodiment, the sequence of the polypeptide comprises or essentially consists of any one of SEQ ID NOs: 62-67 or 76-85.

[0371] In yet another embodiment, the sequence of the polypeptide comprises or essentially consists of SEQ ID NO: 82 or SEQ ID NO: 83.

[0372] The inventors found that polypeptides with these specific sequences were particularly potent and effective in killing HER2-positive tumor cells, even when the cells only expressed HER2 at low levels.

[0373] In another embodiment, the sequence of the polypeptide comprises or essentially consists of SEQ ID NO:

[0374] 82. In an in vivo model this polypeptide showed potent and effective tumor cell killing, observed by a significant reduction in tumor volume compared to baseline. This makes this polypeptide very attractive as a potential treatment for HER2-positive cancer. This is further elaborated on in the Examples section below.

[0375] The components, preferably ISVDs, of said multispecific-multivalent polypeptides described herein may be linked to each other directly or by one or more suitable linkers, such as peptidic linkers.

[0376] The use of linkers to connect two or more (poly)peptides is well known in the art.

[0377] One frequently used class of peptidic linkers are known as the "Gly-Ser" or "GS" linkers. These are linkers that essentially consist of glycine (G) and serine (S) residues, and usually comprise one or more repeats of a peptide motif such as the GGGGS (SEQ ID NO: 130) motif (for example, exhibiting the formula (Gly-Gly- Gly-Gly-Ser)nin which n may be 1, 2, 3, 4, 5, 6, 7 or more). Some often-used examples of such GS linkers are 9GS linkers (GGGGSGGGS, SEQ ID NO: 133), 15GS linkers (n=3) and 35GS linkers (n=7). Reference is for example made to Chen et al. 2013 (Adv. Drug Deliv. Rev. 65(10): 1357-1369) and Klein et al. 2014 (Protein Eng. Des. Sei. 1 (10): 325-330). In the polypeptide(s) disclosed herein, the use of GGS, 5GS and 9GS linkers to link the components of the polypeptide to each other is preferred.

[0378] Examples of suitable linkers are given in Table A-10 below.

[0379] Table A-10: Linker sequences ("ID" refers to the SEQ ID NO as used herein)

[0380] In another embodiment, the polypeptide exhibits reduced binding by pre-existing antibodies present in human serum. To this end, in one embodiment of the present technology, the polypeptide exhibits a valine (V) at amino acid position 11 and a leucine (L) or threonine (T) at amino acid position 89 (according to Kabat numbering) in at least one ISVD (and preferably the ISVD at the C-terminal end of the polypeptide), but preferably in each ISVD.

[0381] ISVDs with these substitutions on positions 11 and 89 are e.g., A032800154 (SEQ ID NO: 33), A032801524 (SEQ ID NO: 34), A032801529 (SEQ ID NO: 35), A032801483 (SEQ ID NO: 36), A032801484 (SEQ ID NO: 37), A032801579 (SEQ ID NO: 38), A032801441 (SEQ ID NO: 39), A032801467 (SEQ ID NO: 40), A032801497 (SEQ ID NO: 41), A032801499(SEQ ID NO: 42), A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801633 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), A032801593 (SEQ ID NO: 53).

[0382] In a further embodiment, the polypeptide exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD. The C- terminus of an ISVD is normally VTVSS (SEQ ID NO: 146).

[0383] In yet another embodiment, the polypeptide exhibits a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD. In another embodiment, the ISVD exhibits a lysine (K) or glutamine (Q) at position 112 (according to Kabat numbering) and / or a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least on ISVD. In these embodiments, the C-terminus of the ISVD is VKVSS (SEQ ID NO: 147), VQVSS (SEQ ID NO: 148), VTVKS (SEQ ID NO: 149), VTVQS (SEQ ID NO: 150), VKVKS (SEQ ID NO: 151), VKVQS (SEQ ID NO: 152), VQVKS (SEQ ID NO: 153), or VQVQS (SEQ ID NO: 154) such that, after addition of a single alanine, the C- terminus of the polypeptide for example exhibits the sequence VTVSSA (SEQ ID NO: 155), VKVSSA (SEQ ID NO: 156), VQVSSA (SEQ ID NO: 157), VTVKSA (SEQ ID NO: 158), VTVQSA (SEQ ID NO: 159), VKVKSA (SEQ ID NO: 160), VKVQSA (SEQ ID NO: 161), VQVKSA (SEQ ID NO: 162), or VQVQSA (SEQ ID NO: 163), preferably VTVSSA.

[0384] In another embodiment of the present technology, the polypeptide exhibits a valine (V) at amino acid position 11 and a leucine (L) at amino acid position 89 (according to Kabat numbering) in at least the C- terminal ISVD, optionally a lysine (K) or glutamine (Q) at position 110 (according to Kabat numbering) in at least one ISVD, and exhibits an extension of 1 to 5 (preferably naturally occurring) amino acids, such as a single alanine (A) extension, at the C-terminus of the C-terminal ISVD (such that the C-terminus of the polypeptide for example consists of the sequence VTVSSA, VKVSSA or VQVSSA, preferably VTVSSA). See e.g. WO 2012 / 175741 and WO 2015 / 173325 for further information in this regard.

[0385] A list of all generated polypeptides is provided below in Table A-ll.

[0386] Table A-ll: Polypeptides of to the present technology

[0387] In one embodiment, the ISVD or polypeptide of the present technology may be linked (optionally via a suitable linker or hinge region) to one or more constant domains (for example, 2 or 3 constant domains that can be used as part of / to form an Fc portion), to an Fc portion and / or to one or more antibody parts, fragments or domains that confer one or more effector functions to the ISVD or polypeptide and / or may confer the ability to bind to one or more Fc receptors. For example, for this purpose, and without being limited thereto, the one or more further amino acid sequences may comprise one or more CH2 and / or CHS domains of an antibody, such as from a heavy chain antibody (as described herein) and more preferably from a conventional human 4-chain antibody; and / or may form (part of) and Fc region, for example from IgG (e.g. from IgGl, lgG2, lgG3 or lgG4), from IgE or from another human Ig such as IgA, IgD or IgM. For example, WO 94 / 04678 describes heavy chain antibodies comprising a Camelid VHH domain or a humanized derivative thereof in which the Camelidae CH2 and / or CHS domain have been replaced by human CH2 and CHS domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a VH H and human CH2 and CHS domains (but no CHI domain), which immunoglobulin has the effector function provided by the CH2 and CHS domains and which immunoglobulin can function without the presence of any light chains. Other amino acid sequences that can be suitably linked to the ISVD or polypeptide of the present technology so as to provide an effector function will be clear to the skilled person and may be chosen on the basis of the desired effector function(s). Reference is for example made to WO 04 / 058820, WO 99 / 42077, WO 02 / 056910 and WO 05 / 017148, as well as the review by Hol liger and Hudson, supra; and to WO 09 / 068628. Coupling of ISVD or polypeptide to an Fc portion may also lead to an increased half-life, compared to the corresponding ISVD or polypeptide. For some applications, the use of an Fc portion and / or of constant domains (i.e. CH2 and / or CHS domains) that confer increased halflife without any biologically significant effector function may also be suitable or even preferred. Other suitable constructs comprising one or more ISVDs or polypeptides and one or more constant domains with increased half-life in vivo will be clear to the skilled person and may for example comprise ISVDs or polypeptides linked to a CHS domain, optionally via a linker sequence. Generally, any fusion protein or derivatives with increased half-life will preferably have a molecular weight of more than 50 kD, the cut-off value for renal absorption. 3 Polypeptide constructs

[0388] The present inventors found that combining ISVDs of the present technology with an Fc region resulted in potent constructs that could effectively target HER2-positive tumor cells and eliminate them.

[0389] In one aspect, the present technology relates to a polypeptide construct comprising

[0390] (i) a polypeptide comprising a first ISVD capable of specifically binding to human and / or non-human primate HER2 according to the present technology;

[0391] (ii) a second ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell; and

[0392] (iii) a Fc region which is linked to the polypeptide via a suitable linker or hinge region.

[0393] In another aspect, the present technology relates to a polypeptide construct comprising

[0394] (i) a polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2 according to the present technology;

[0395] (ii) a third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell; and

[0396] (iii) a Fc region which is linked to the polypeptide via a suitable linker or hinge region.

[0397] As used herein, the term "Fc domain" or "Fc region" (used interchangeably) is defined as the portion of a heavy chain constant region beginning in the hinge region just upstream of the papain cleavage site (i.e., residue 216 in IgG, taking the first residue of heavy chain constant region to be 114) and ending at the C- terminus of the antibody. Accordingly, a complete Fc domain comprises at least a hinge domain, a CH2 domain, and a CHS domain.

[0398] In an embodiment, a first and / or second Fc constant domain is derived from an immunoglobulin class selected from a group consisting of: IgM, IgG, IgD, IgA, IgE. Chimeric Fc domains comprising portions of Fc domains from different species or Ig classes can also be employed. In certain aspects, a Fc constant domain is an IgG Fc domain, an IgGl Fc domain, or an lgG4 Fc domain.

[0399] In an embodiment, the Fc region comprises at least one or more parts, fragments, amino acid stretches or domains of the Fc portion of IgG. Techniques for making multispecific binding proteins (e.g., multispecific antibodies) include, but are not limited to, recombinant co-expression of two immunoglobulin heavy chain-light chain pairs having different specificities (see Milstein, C. and Cuello, A. C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific binding proteins may also be made by engineering electrostatic steering effects for making binding protein Fc-heterodimeric molecules (WO 2009 / 089004); cross-linking two or more antibodies orfragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M. et al., Science 229 (1985) 81-83); using leucine zippers to produce bi-specific antibodies (see, e.g., Kostelny, S. A. et al., J. Immunol. 148 (1992) 1547-1553); using "diabody" technology for making multispecific binding protein fragments (see, e.g., Hol liger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and using single-chain Fv (scFv) dimers (see, e.g., Gruber, M et al., J. Immunol. 152 (1994) 5368-5374); and preparing trispecific binding proteins as described, e.g., in Tutt, A. et al., J. Immunol. 147 (1991) 60-69).

[0400] A wide variety of recombinant multispecific binding protein formats have been developed, e.g., by fusion of, e.g., an IgG binding protein format and single chain domains (see Kontermann RE, mAbs 4:2, (2012) 1- 16). Multispecific binding proteins wherein the variable domains VL and VH or the constant domains CL and CHI are replaced by each other are described in WO 2009 / 080251 and WO 2009 / 080252.

[0401] An approach to circumvent the problem of mispaired byproducts, is known as "knob-into-holes". This approach aims at forcing the pairing of two different binding protein heavy chains by introducing mutations into the CH3 domains to modify the contact interface. On one chain, bulky amino acids can be replaced by amino acids with short side chains to create a "hole." Conversely, amino acids with large side chains can be introduced into the other CH3 domain, to create a "knob." By co-expressing these two heavy chains (and two identical light chains, which have to be appropriate for both heavy chains), high yields of heterodimer formation ("knob-hole") versus homodimer formation ("hole-hole" or "knob-knob") was observed (Ridgway JB, Presta LG, Carter P; and WO 1996 / 027011). The percentage of heterodimer could be further increased by remodeling the interaction surfaces of the two CH3 domains using a phage display approach and the introduction of a disulfide bridge to stabilize the heterodimers (Merchant A.M, et al, Nature Biotech 16 (1998) 677-681; Arwell S, Ridgway JB, Wells JA, Carter P., J Mol Biol 270 (1997) 26-35). New approaches for the knob-into-holes technology are described in e.g., in EP 1870459A1. Xie, Z., et al, J Immunol Methods 286 (2005) 95-101 refers to a format of multispecific binding protein using scFvs in combination with knob-into-holes technology for the Fc part. In an embodiment, a polypeptide construct of the present technology comprises a first and a second IgG Fc domain polypeptide that dimerize to form the polypeptide construct. In certain aspects, the first and second IgG Fc domain polypeptides dimerize by knob-into-holes interactions.

[0402] In another embodiment, the first IgG Fc domain polypeptide comprises a knob substitution, and the second IgG Fc domain polypeptide comprises a hole substitution.

[0403] In a further embodiment, the polypeptide construct comprises a knob substitution that is selected from the group consisting of arginine (R), phenylalanine (F), tyrosine (Y), and tryptophan (W).

[0404] In yet another embodiment, the polypeptide construct comprises a hole substitution that is selected from the group consisting of alanine (A), asparagine (N), aspartic acid (D), glycine (G), serine (S), threonine (T), and valine (V).

[0405] In another embodiment, the Fc region comprises or consists of an lgG4 knob-and-hole Fc with FALA mutations.

[0406] "FALA" has its regular scientific meaning and means the substitution of a phenylalanine (F) with an alanine (A) and a leucine (L) with an alanine (A) at neighboring positions within the Fc domain. A FALA mutation in the Fc region, that has been previously described (Alegre ML, Peterson LJ, et al. Transplantation 57: 1537-43(1994)), eliminates binding to all Fc receptors, except FcRn. The lgG4 knob-and-hole Fc with FALA mutations is an otherwise inactive Fc domain that still provides the polypeptide construct with an increased half-life.

[0407] In an embodiment, the second or third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell is at the N-terminal position of the hole chain of the Fc region.

[0408] In another embodiment, the second or third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell has a CDR1 with amino acid sequence XIX2VHKIN FX3G (SEQ ID NO: 214), a CDR2 with amino acid sequence HISIGDQTD (SEQ ID NO: 215) and a CDR3 with amino acid sequence X4SRIX5PYDY (SEQ ID NO: 216), according to Abm definition, wherein

[0409] - Xi is selected from G, W, E and D;

[0410] - X2is selected from D, Y G and W;

[0411] - X3is selected from L and Y;

[0412] - X4is selected from F and L; and

[0413] - X5is selected from W, Y, T, S and Q.

[0414] In some embodiments, the second or third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 219.

[0415] In some embodiments, the second or third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY).

[0416] In some embodiments, the second or third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIWPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 259.

[0417] In some embodiments, the second or third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell is an ISVD comprising a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIWPYDY).

[0418] In the polypeptide construct of the present technology, the first and second ISVDs capable of specifically binding to human and / or non-human primate HER2 according to the present technology can be at the C- terminal end of the hole chain of the Fc region or at the N-terminal or the C-terminal end of the knob chain of the Fc region. For example, in some embodiments, the first and second ISVDs capable of specifically binding to human and / or non-human primate HER2 according to the present technology are at the C-terminal end of the hole chain of the Fc region, or at the C-terminal end of the knob chain of the Fc region, or at the N-terminal end of the knob chain of the Fc region.

[0419] In another embodiment, the first and second ISVD capable of specifically binding to human and / or non- human primate HER2 according to the present technology are at the N-terminal position of the knob chain of the Fc region.

[0420] In a further embodiment, said first and second ISVD capable of specifically binding to human and / or non- human primate HER2 according to the present technology essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO:1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XSIX6RVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein

[0421] Xi is selected from I and S; X2is selected from V, R and Y;

[0422] X3is selected from A and R;

[0423] X4is selected from I, L and M;

[0424] X5is selected from G and L;

[0425] X6is selected from D and S;

[0426] X7is selected from E and G;

[0427] X8is selected from D, E and V, or e. the amino acid sequence of CDR1 is X9IPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIXi2SXi3GXi4PXi5 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is VNG DXM (SEQ ID NO: 254), according to AbM definition, wherein

[0428] X3is selected from A and R;

[0429] X4is selected from I, L and M;

[0430] X9is selected from G and R;

[0431] Xio is selected from F and A;

[0432] Xu is selected from T and I;

[0433] Xi2is selected from R and G;

[0434] Xi3is selected from absence of an amino acid residue and S;

[0435] X14 is selected from A and T;

[0436] Xis is selected from V and A;

[0437] Xie is selected from I and Y; or f. the amino acid sequence of CDR1 is X9IPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TXi7Xi2SXi3GXi4PXi5 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is VNGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0438] X3is selected from A and R;

[0439] X4is selected from I, L and M;

[0440] X9is selected from G and R;

[0441] Xio is selected from F and A;

[0442] Xu is selected from T and I;

[0443] Xi2is selected from R and G;

[0444] Xi3is selected from absence of an amino acid residue or S;

[0445] X14 is selected from A and T;

[0446] Xis is selected from V, Y and A; Xi6 is selected from I and Y;

[0447] X17 is selected from I and S; and wherein the first and second ISVD may have the same or a different sequence.

[0448] In an embodiment, the first and second ISVD capable of specifically binding to human and / or non-human primate HER2 according to the present technology have CDR sequences selected from the following: a. the amino acid sequence of CDR1 is SEQ ID NO: 1, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 13-16, and the amino acid sequence of CDR3 is SEQ ID NO: 3, according to AbM definition; or b. the amino acid sequence of CDR1 is selected from SEQ ID NOs: 17-20, the amino acid sequence of CDR2 is SEQ ID NO: 5, and the amino acid sequence of CDR3 is SEQ ID NO: 6, according to AbM definition; or c. the amino acid sequence of CDR1 is SEQ ID NO: 7, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 21, 23 and 24, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 22 and 25; according to AbM definition; or d. the amino acid sequence of CDR1 is SEQ ID NO: 10, the amino acid sequence of CDR2 is SEQ ID NO: 11, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 26-28; according to AbM definition.

[0449] In another embodiment, the first and second ISVD capable of specifically binding to human and / or non- human primate HER2 according to the present technology have CDR sequences selected from the following: a. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 13, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or b. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 14, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or c. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 15, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or d. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 16, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or e. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 17, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or f. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 18, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or g. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 19, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or h. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 20, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or i. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or j. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or k. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or l. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or m. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or n. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or o. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 26; according to AbM definition; or p. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 27; according to AbM definition; or q. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 28; according to AbM definition.

[0450] In some embodiments, the first and second ISVD capable of specifically binding to human and / or nonhuman primate HER2 according to the present technology essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR1, CDR2 and CDR3 sequence selected from the CDR1, CDR2 and CDR3 sequences presented in Table A-4. In another embodiment, the first and second ISVD capable of specifically binding to human and / or nonhuman primate HER2 according to the present technology essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein the first and / or second ISVD has CDR1, CDR2 and CDR3 sequence selected from the combination of CDR sequences presented in the same row in Table A-4.

[0451] In an embodiment, the first and second ISVD capable of specifically binding to human and / or non-human primate HER2 according to the present technology comprise (in addition to the CDRs as defined above) one or more, or all, of the framework regions as indicated in Table A-5.

[0452] In another embodiment, the first and second ISVD capable of specifically binding to human and / or non- human primate HER2 according to the present technology comprise or essentially consist of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0453] In another embodiment, the first and second ISVD capable of specifically binding to human and / or non- human primate HER2 according to the present technology comprise or essentially consist of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%.

[0454] In one embodiment, the first and second ISVD capable of specifically binding to human and / or non-human primate HER2 according to the present technology comprise or consist of the full amino acid sequence of any one of A032800154 (SEQ ID NO: 33), A032801524 (SEQ ID NO: 34), A032801529 (SEQ ID NO: 35), A032801483 (SEQ ID NO: 36), A032801484 (SEQ ID NO: 37), A032801579 (SEQ ID NO: 38), A032801441 (SEQ ID NO: 39), A032801467 (SEQ ID NO: 40), A032801497 (SEQ ID NO: 41), A032801499 (SEQ ID NO: 42), A032801618 (SEQ ID NO: 43), A032801621 (SEQ ID NO: 44), A032801623 (SEQ ID NO: 45), A032801663 (SEQ ID NO: 46), A032801668 (SEQ ID NO: 47), A032801613 (SEQ ID NO: 48), A032801689 (SEQ ID NO: 49), A032801690 (SEQ ID NO: 50), A032801588 (SEQ ID NO: 51), A032801589 (SEQ ID NO: 52), or A032801593 (SEQ ID NO: 53) (Table A-3). In yet a further embodiment, in the polypeptide construct of the present technology, a. the first ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and b. the second ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and c. the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 and SEQ ID NO: 56.

[0455] In yet a further embodiment, in the polypeptide construct of the present technology, a. the first ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and b. the second ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and c. the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 and SEQ ID NO: 268.

[0456] In another embodiment, in the polypeptide construct of the present technology, a. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or b. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or c. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second

[0457] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or d. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second

[0458] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or e. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or f. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or g. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 40, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or h. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or i. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or j. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or k. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or l. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 51, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or m. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 53, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or n. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 45, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or o. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56.

[0459] In another embodiment, in the polypeptide construct of the present technology, a. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or b. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, the second

[0460] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or c. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second

[0461] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or d. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or e. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or f. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second

[0462] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or g. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 40, the second

[0463] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or h. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second

[0464] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or i. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second

[0465] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or j. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or k. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or l. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 51, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or m. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 53, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or n. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 45, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or o. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268.

[0466] The inventors found that the combinations of these particular ISVDs with an Fc format resulted in constructs that had particular good potency and efficacy in targeting HER2-positive cells even when the cells expressed HER2 at low levels. Additionally, thanks to the Fc-region, the constructs could remain in circulation for an extended time.

[0467] In another embodiment, the polypeptide construct has an affinity for human and / or non-human primate HER2 of between 1-600 nM, preferably between 1-500 nM, more preferably between 1-100 nM, most preferably between 1-10 nM.

[0468] In an embodiment, the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NOs: 68, 86, 88-92 or 94-97. In another embodiment, the sequence of the knob chain of the Fc region comprises or essentially consists of any one of SEQ ID NOs: 69-74, 87 or 93.

[0469] In yet another embodiment, in the polypeptide construct of the present technology, a. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 69; or b. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 70; or c. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 71; or d. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 72; or e. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 73; or f. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 74; or g. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 86 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or h. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0470] 88 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or i. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0471] 89 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or j. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0472] 90 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or k. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0473] 91 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or l. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0474] 92 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or m. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0475] 94 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or n. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0476] 95 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or o. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0477] 96 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or p. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0478] 97 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93.

[0479] In another embodiment, the sequence of the polypeptide construct comprises or essentially consists of the hole chain sequence of SEQ ID NO: 92 and the knob chain sequence of SEQ ID NO: 93 or the hole chain sequence of SEQ ID NO: 97 and the knob chain sequence of SEQ ID NO: 93.

[0480] The inventors found that out of all tested compounds, these combinations of knob- and hole-chain were the most potent and had the least undesirable effects.

[0481] In a further embodiment, the sequence of the polypeptide construct comprises or essentially consists of the hole chain sequence of SEQ ID NO: 97 and the knob chain sequence of SEQ ID NO: 93. When this polypeptide construct was tested in an in vivo model it was highly potent and showed a relatively low safety risk. This makes this polypeptide construct very suitable for use in treatment of HER2- positive cancers. This is further outlined in the Examples section below.

[0482] A list of all generated polypeptide constructs is provided below in Table A-12.

[0483] Table A-12: Polypeptide constructs of the present technology ("ID" refers to the given SEQ ID NO) 4 Nucleic acid molecules

[0484] Also provided is a nucleic acid molecule encoding the ISVDs, polypeptides or polypeptide constructs as disclosed herein.

[0485] A "nucleic acid molecule" (used interchangeably with "nucleic acid") is a chain of nucleotide monomers linked to each other via a phosphate backbone to form a nucleotide sequence. A nucleic acid may be used to transform / transfect a host cell or host organism, e.g. for expression and / or production of a polypeptide. Suitable hosts or host cells for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. A (non-human) host or host cell comprising a nucleic acid encoding the polypeptide is also encompassed by the technology.

[0486] A nucleic acid may be for example DNA, RNA, or a hybrid thereof, and may also comprise (e.g. chemically) modified nucleotides, like PNA. It can be single- or double-stranded and is preferably in the form of doublestranded DNA. For example, the nucleotide sequences may be genomic DNA or cDNA.

[0487] The nucleic acids can be prepared or obtained in a manner known per se, and / or can be isolated from a suitable natural source. Nucleotide sequences encoding naturally occurring (poly)peptides can for example be subjected to site-directed mutagenesis, so as to provide a nucleic acid molecule encoding polypeptide with sequence variation. Also, as will be clear to the skilled person, to prepare a nucleic acid, also several nucleotide sequences, such as at least one nucleotide sequence encoding a targeting moiety and for example nucleic acids encoding one or more linkers can be linked together in a suitable manner.

[0488] Techniques for generating nucleic acids will be clear to the skilled person and may for instance include, but are not limited to, automated DNA synthesis; site-directed mutagenesis; combining two or more naturally occurring and / or synthetic sequences (or two or more parts thereof), introduction of mutations that lead to the expression of a truncated expression product; introduction of one or more restriction sites (e.g. to create cassettes and / or regions that may easily be digested and / or ligated using suitable restriction enzymes), and / or the introduction of mutations by means of a PCR reaction using one or more "mismatched" primers. 5 Vectors

[0489] Also provided is a vector comprising the nucleic acid molecule encoding the ISVDs, polypeptides or polypeptide constructs as disclosed herein.

[0490] A "vector" as used herein is a vehicle suitable for carrying genetic material into a cell. A vector includes naked nucleic acids, such as plasmids or mRNAs, or nucleic acids embedded into a bigger structure, such as liposomes or viral vectors.

[0491] Vectors generally comprise at least one nucleic acid that is optionally linked to one or more regulatory elements, such as for example one or more suitable promoter(s), enhancer(s), terminator(s), etc.). The vector preferably is an expression vector, i.e. a vector suitable for expressing an encoded polypeptide or construct under suitable conditions, e.g. when the vector is introduced into a (e.g. human) cell. For DNA- based vectors, this usually includes the presence of elements for transcription (e.g. a promoter and a polyA signal) and translation (e.g. Kozak sequence).

[0492] Preferably, in the vector, said at least one nucleic acid and said regulatory elements are "operably linked" to each other, by which is generally meant that they are in a functional relationship with each other. For instance, a promoter is considered "operably linked" to a coding sequence if said promoter is able to initiate or otherwise control / regulate the transcription and / or the expression of a coding sequence (in which said coding sequence should be understood as being "under the control of" said promotor). Generally, when two nucleotide sequences are operably linked, they will be in the same orientation and usually also in the same reading frame. They will usually also be essentially contiguous, although this may also not be required.

[0493] Preferably, any regulatory elements of the vector are such that they are capable of providing their intended biological function in the intended host cell or host organism.

[0494] For instance, a promoter, enhancer or terminator should be "operable" in the intended host cell or host organism, by which is meant that for example said promoter should be capable of initiating or otherwise controlling / regulating the transcription and / or the expression of a nucleotide sequence - e.g. a coding sequence - to which it is operably linked. 6 Compositions

[0495] The technology also provides a composition comprising at least one ISVD, polypeptide or polypeptide construct as disclosed herein, at least one nucleic acid molecule encoding such an ISVD, polypeptide or polypeptide construct as disclosed herein or at least one vector comprising such a nucleic acid molecule.

[0496] The composition may be a pharmaceutical composition. The composition may further comprise at least one pharmaceutically acceptable carrier, diluent or excipient and / or adjuvant, and optionally comprise one or more further pharmaceutically active polypeptides and / or compounds.

[0497] 7 Host organisms

[0498] The technology also pertains to (non-human) hosts, host organisms or host cells comprising the ISVDs, polypeptides or polypeptide constructs as disclosed herein, the nucleic acid encoding the ISVDs or polypeptides as disclosed herein, and / or the vector comprising the nucleic acid molecule encoding the ISVDs or polypeptides as disclosed herein.

[0499] Suitable host cells or host organisms are clear to the skilled person, and are for example any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.

[0500] 8 Methods and uses

[0501] The technology also provides a method for producing the ISVDs, polypeptides or polypeptide constructs as disclosed herein. The method may comprise transforming / transfecting a (non-human) host, host organism or host cell with a nucleic acid encoding the ISVD, polypeptide or polypeptide construct, expressing the ISVD, polypeptide or polypeptide construct in the host, optionally followed by one or more isolation and / or purification steps. Specifically, the method may comprise: a. expressing, in a suitable host, host cell or (non-human) host organism or in another suitable expression system, a nucleic acid sequence encoding the ISVD, polypeptide or polypeptide construct; optionally followed by: b. isolating and / or purifying the ISVD, polypeptide or polypeptide construct. Suitable hosts, host cells or (non-human) host organisms for production purposes will be clear to the skilled person, and may for example be any suitable fungal, prokaryotic or eukaryotic cell or cell line or any suitable fungal, prokaryotic or eukaryotic organism. Specific examples include HEK293 cells, CHO cells, Escherichia coli or Pichia pastoris. The most preferred host is Pichia pastoris.

[0502] Typically, the polypeptides and polypeptide constructs of the current technology combine high affinity HER2 recognition on the target cell with T cell activation, resulting in an activation that is independent of the T cells' natural specificity. The first and second ISVD of the polypeptide or polypeptide construct of the present technology has high affinity for / specifically binds to HER2 on a target cell and the third ISVD has high affinity for / specifically binds to an effector cell, preferably the TCR of said effector cell, and even more preferably the constant domain of the TCR.

[0503] An effector cell is a cell comprising a TCR complex, preferably an immune cell, such as a T cell, preferably a CD4+ T-helper cell (also known as CD4 cell, T-helper cell or T4 cell), more preferably a cytotoxic T cell (also known as TC cell, CTL or CD8+ T cells) or Natural Killer T cells (NKT cells). In some embodiments, the cell is present in vivo. In some embodiments, the cell is present in vitro. The effector cell of the present technology relates in particular to mammalian cells, preferably to primate cells, and even more preferably to human cells.

[0504] "T cell activation" as used herein refers to one or more cellular response(s) of a T cell, e.g. a cytotoxic T cell, such as selected from: proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, expression of activation markers, and redirected target cell lysis. The polypeptides and polypeptide constructs of the present technology are capable of inducing T cell activation. Suitable assays to measure T cell activation are known in the art described herein, for instance as described in WO 99 / 54440 or by Schlereth et al. 2005 (Cancer Immunol. Immunother. 20: 1-12), or as exemplified in the examples or below.

[0505] In an embodiment, the present technology relates to a polypeptide or polypeptide construct as described herein, wherein said polypeptide or polypeptide construct induces T cell activation. Preferably, the polypeptide or polypeptide construct of the present technology induces T cell activation only when said first and second ISVD are bound to HER2 on a target cell. In another embodiment, the present technology relates to a polypeptide or polypeptide construct as described herein, wherein said T cell activation depends on presenting said polypeptide or polypeptide construct bound to said HER2 on a target cell to a T cell.

[0506] T cell activation by the polypeptides of the present technology can be monitored by upregulation of CD69, CD25 and various cell adhesion molecules, de novo expression and / or release of cytokines (e.g., IFN-y, TNF-a, IL-6, IL-2, IL-4 and IL-10), upregulation of granzyme and perforin expression, and / or cell proliferation, membrane blebbing, activation of procaspases 3 and / or 7, fragmentation of nuclear DNA and / or cleavage of caspase substrate poly (ADPribose) polymerase. Preferably, redirected lysis of target cells by multispecific-multivalent polypeptides is independent of T cell receptor specificity, presence of MHC class I and / or 02 microglobulin, and / or of any co-stimulatory stimuli.

[0507] In a further embodiment, the present technology relates to a polypeptide or polypeptide construct as described herein, wherein said T cell activation is independent from MHC recognition.

[0508] In another embodiment, the present technology relates to a polypeptide or polypeptide construct as described herein, wherein said T cell activation causes one or more cellular response of said T cell, wherein said cellular response is selected from the group consisting of proliferation, differentiation, cytokine secretion, cytotoxic effector molecule release, cytotoxic activity, expression of activation markers and redirected target cell lysis.

[0509] As used herein, the term "potency" is a measure of the biological activity of an agent, such as a polypeptide or an ISVD. Potency of an agent can be determined by any suitable method known in the art, such as for instance as described in the experimental section. Cell culture-based potency assays are often the preferred format for determining biological activity since they measure the physiological response elicited by the agent and can generate results within a relatively short period of time. Various types of cell based assays, based on the mechanism of action of the product, can be used, including but not limited to proliferation assays, cytotoxicity assays, cell killing assays, reporter gene assays, cell surface receptor binding assays, and assays to measure induction / inhibition of functionally essential proteins or other signal molecules (such as phosphorylated proteins, enzymes, cytokines, cAMP and the like), Ramos B cell depletion model, T cell mediated tumor cell killing assay (for instance as set out in the Examples section), all well known in the art. The "efficacy" measures the maximum strength of the effect itself, at saturating polypeptide (construct) concentrations. Efficacy indicates the maximum response achievable by the polypeptide or polypeptide construct of the present technology. It refers to the ability of a polypeptide (construct) to produce the desired (therapeutic) effect.

[0510] Accordingly, in an embodiment, the present technology relates to a polypeptide or polypeptide construct as described herein, wherein said T cell activation causes inhibition of an activity of said target cell, such as to delay or minimize the spread of the target cell, to inhibit or delay growth and / or proliferation of the target cell, and / or to kill the target cell (e.g., cause regression of the disorder and / or symptoms), by more than about 10%, more than 10%, such as 20%, 30%, or 40%, or even more than 50%, such as more than 60%, such as 70%, 80% or even more than 90%, such as 100%. In a particular embodiment, the T cell activation causes lysis of the target cell, by more than about 10%, more than 10%, such as 20%, 30%, or 40%, or even more than 50%, such as more than 60%.

[0511] In another embodiment, the polypeptide or polypeptide construct as described herein causes a human T cell to lyse the target cell with an IC50 value selected from the group consisting of at most about 10'9M, at most about 1010M, and at most about 1011M, said IC50 value as determined in a T cell mediated killing assay.

[0512] In a further embodiment, the polypeptide or polypeptide construct as described herein cause a nonhuman primate T cell to lyse the target cell with an IC50 value selected from the group consisting of at most about 10'9M, and at most about 1010M, and at most about 1011M, such as with an IC50 value of 10'9M or lower (better affinity), 1010M or lower (better affinity), or even 1011M or lower (better affinity), said IC50 value as determined in a T cell mediated killing assay (such as e.g., described herein).

[0513] The ISVD, polypeptide, polypeptide construct, nucleic acid molecule or vector as described, or the composition comprising the ISVD, polypeptide or polypeptide construct, nucleic acid molecule or vector, are useful as a medicament. Accordingly, the technology provides the ISVD, polypeptide, polypeptide construct, nucleic acid molecule or vector as described, or a composition comprising the ISVD, polypeptide or polypeptide construct, nucleic acid molecule or vector for use as a medicament.

[0514] Additionally, provided is the ISVD, polypeptide, polypeptide construct, nucleic acid molecule or vector as described, or a composition comprising the ISVD, polypeptide or polypeptide construct, nucleic acid molecule or vector for use in the treatment of cancer. Preferably, said cancer is a HER2 positive cancer, wherein said HER2 cancer is preferably selected from gastric cancer, gastroesophageal junction adenocarcinoma or breast cancer.

[0515] Additionally, provided is a method of treating cancer, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of the ISVD, polypeptide, polypeptide construct, nucleic acid molecule or vector as described herein, or a composition comprising the ISVD, polypeptide or polypeptide construct, nucleic acid molecule or vector. Preferably, said cancer is a HER2 positive cancer, wherein said HER2 cancer is preferably selected from gastric cancer, gastroesophageal junction adenocarcinoma or breast cancer.

[0516] Further provided is the use of the ISVD, polypeptide, polypeptide construct, nucleic acid molecule or vector as described herein, or a composition comprising the ISVD, polypeptide, polypeptide construct, nucleic acid molecule or vector in the preparation of a medicament.

[0517] A "subject" as referred to in the context of the technology can be any animal, preferably a mammal. Among mammals, a distinction can be made between humans and non-human mammals. Non-human animals may be for example companion animals (e.g. dogs, cats), livestock (e.g. bovine, equine, ovine, caprine, or porcine animals), or animals used generally for research purposes and / or for producing antibodies (e.g. mice, rats, rabbits, cats, dogs, goats, sheep, horses, pigs, non-human primates, such as cynomolgus monkeys, or camelids, such as llama or alpaca).

[0518] In the context of prophylactic and / or therapeutic purposes, the subject can be any animal, and more specifically any mammal, but preferably is a human subject. As used herein, the terms "treat", "treatment" and "treating" in the context of administering (a) therapy(ies) to a subject refer to the reduction or amelioration of the progression, severity, and / or duration of a disorder associated with a hyperprol iterative cell disorder, e.g., cancer, and / or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies (including, but not limited to, the administration of one or more prophylactic or therapeutic agents). In specific embodiments, the terms "treat", "treatment" and "treating" in the context of administering a therapy / therapies to a subject refer to the reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., cancer, refers to a reduction in cancer cells by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100% relative to a control (e.g., a negative control such as phosphate buffered saline). In other embodiments, the terms "treat", "treatment" and "treating" in the context of administering a therapy, or therapies, to a subject refer to the reduction or amelioration of the progression, severity, and / or duration of a hyperproliferative cell disorder, e.g., cancer, refers to no change in cancer cell number, a reduction in hospitalization time, a reduction in mortality, or an increase in survival time of the subject with cancer.

[0519] Substances (including ISVDs, polypeptides, polypeptide constructs, nucleic acid molecules and vectors) or compositions may be administered to a subject by any suitable route of administration, for example by enteral (such as oral or rectal) or parenteral (such as epicutaneous, sublingual, buccal, nasal, intra-articular, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, transdermal, or transmucosal) administration. Parenteral administration, such as intramuscular, subcutaneous or intradermal, administration is preferred. Most preferred is subcutaneous administration.

[0520] An effective amount of an ISVD, polypeptide, polypeptide construct, a nucleic acid molecule or vector as described, or a composition comprising the ISVD, polypeptide or polypeptide construct, nucleic acid molecule or vector can be administered to a subject in order to provide the intended treatment results.

[0521] One or more doses can be administered. If more than one dose is administered, the doses can be administered in suitable intervals in order to maximize the effect of the ISVD, polypeptide, polypeptide construct, composition, nucleic acid molecule or vector. The present technology will be further illustrated by, but not limited to, the Embodiments and Examples set out below.

[0522] In Table A-13 below, a list of all sequences can be found.

[0523] Table A-13: List of sequences

[0524] EMBODIMENTS

[0525] Embodiment 1 Immunoglobulin single variable domain (ISVD) which specifically binds to human and / or non-human primate HER2, wherein said ISVD essentially consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO:1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XSIX6R G TS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein

[0526] Xi is selected from I or S;

[0527] X2is selected from V, R or Y;

[0528] X3is selected from A or R;

[0529] X4is selected from I, L or M;

[0530] X5is selected from G or L;

[0531] X6is selected from D or S;

[0532] X7is selected from E or G;

[0533] X8is selected from D, E or V; or e. the amino acid sequence of CDR1 is XglPXioSXnX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIX12SX13GX14PX15 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is VNGDXM (SEQ ID NO: 254), according to AbM definition, wherein

[0534] Xi is selected from I or S;

[0535] X2is selected from V, R or Y;

[0536] X3is selected from A or R;

[0537] X4is selected from I, L or M;

[0538] X5is selected from G or L; X6is selected from D or S;

[0539] X7is selected from E or G;

[0540] X8is selected from D, E or V;

[0541] X9is selected from G or R;

[0542] Xio is selected from F or A;

[0543] Xu is selected from T or I;

[0544] X12 is selected from R or G;

[0545] X13 is selected from absence of an amino acid residue or S;

[0546] X14 is selected from A or T;

[0547] X15 is selected from V or A;

[0548] Xie is selected from I or Y; or f. the amino acid sequence of CDR1 is X9lPXioSXnX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TX17X12SX13GX14PX15 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is NGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0549] X3 is selected from A and R;

[0550] X4is selected from I, L and M;

[0551] X9is selected from G and R;

[0552] Xio is selected from F and A;

[0553] Xu is selected from T and I;

[0554] X12 is selected from R and G;

[0555] X13 is selected from absence of an amino acid residue or S;

[0556] X14 is selected from A and T;

[0557] X15 is selected from V, Y and A;

[0558] Xie is selected from I and Y;

[0559] X17 is selected from I and S; and wherein the full amino acid sequence of the ISVD is different from SEQ ID NOs: 29-32.

[0560] Embodiment 2 ISVD according to embodiment 1, wherein a. the amino acid sequence of CDR1 is SEQ ID NO: 1, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 13-16, and the amino acid sequence of CDR3 is SEQ ID NO: 3, according to AbM definition; or b. the amino acid sequence of CDR1 is selected from SEQ ID NOs: 17-20, the amino acid sequence of CDR2 is SEQ ID NO: 5, and the amino acid sequence of CDR3 is SEQ ID NO: 6, according to AbM definition; or c. the amino acid sequence of CDR1 is SEQ ID NO: 7, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 21, 23 and 24, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 22 and 25; according to AbM definition; or d. the amino acid sequence of CDR1 is SEQ ID NO: 10, the amino acid sequence of CDR2 is SEQ ID NO: 11, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 26-28; according to AbM definition.

[0561] Embodiment 3 ISVD according to embodiment 1 or embodiment 2, wherein a. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 13, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or b. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 14, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or c. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 15, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or d. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 16, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or e. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 17, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or f. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 18, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or g. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 19, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or h. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 20, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or i. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or j. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or k. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or l. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or m. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or n. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or o. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 26; according to AbM definition; or p. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 27; according to AbM definition; or q. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 28; according to AbM definition.

[0562] Embodiment 4 ISVD according to any one of embodiments 1-3, wherein the ISVD is a heavy-chain ISVD.

[0563] Embodiment 5 ISVD according to any one of embodiments 1-4, wherein the ISVD is selected from a VHH, a humanized VHH, a camelized VH, a domain antibody, a single domain antibody, and a dAb.

[0564] Embodiment 6 ISVD according to any one of embodiments 1-5, wherein the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0565] Embodiment 7 ISVD according to any one of embodiments 1-6, wherein the ISVD comprises or consists of a sequence selected from SEQ ID NOs: 33-53.

[0566] Embodiment 8 Polypeptide comprising an ISVD according to any one of embodiments 1-7. Embodiment 9 Polypeptide comprising a first and second ISVD capable of specifically binding to human and / or non-human primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively, wherein the first and / or second ISVD has CDR sequences selected from the following: a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO: 1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XSIX6RVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, wherein

[0567] Xi is selected from I and S;

[0568] X2 is selected from V, R and Y;

[0569] X3is selected from A and R;

[0570] X4is selected from I, L and M;

[0571] X5is selected from G and L;

[0572] X6is selected from D and S;

[0573] X7is selected from E and D;

[0574] X8is selected from D, E and V; or e. the amino acid sequence of CDR1 is X9lPXioSXiiX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TIXi2SXi3GXi4PXi5 (SEQ ID NO: 253), and the amino acid sequence of CDR3 is VNGDXM (SEQ ID NO: 254), according to AbM definition, wherein

[0575] Xi is selected from I or S;

[0576] X2is selected from V, R or Y;

[0577] X3is selected from A or R;

[0578] X4is selected from I, L or M; X5is selected from G or L;

[0579] X6is selected from D or S;

[0580] X7is selected from E or G;

[0581] X8is selected from D, E or V;

[0582] X9is selected from G or R;

[0583] Xio is selected from F or A;

[0584] Xu is selected from T or I;

[0585] X12 is selected from R or G;

[0586] X13 is selected from absence of an amino acid residue or S;

[0587] X14 is selected from A or T;

[0588] X15 is selected from V or A:

[0589] XM is selected from I or Y; f. the amino acid sequence of CDR1 is X9lPXioSXnX3TX4A (SEQ ID NO: 252), the amino acid sequence of CDR2 is TX17X12SX13GX14PX15 (SEQ ID NO: 260), and the amino acid sequence of CDR3 is NGDXie (SEQ ID NO: 254), according to AbM definition, wherein

[0590] X3is selected from A and R;

[0591] X4is selected from I, L and M;

[0592] X9is selected from G and R;

[0593] Xio is selected from F and A;

[0594] Xu is selected from T and I;

[0595] X12 is selected from R and G;

[0596] X13 is selected from absence of an amino acid residue or S;

[0597] X14 is selected from A and T;

[0598] X15 is selected from V, Y and A;

[0599] XM is selected from I and Y;

[0600] X17 is selected from I and Sand wherein the second ISVD has a sequence that may be the same or different from the first ISVD.

[0601] Embodiment 10 Polypeptide according to embodiment 9, wherein the first and second ISVD have sequences that are selected from sequences that have a degree of sequence identity with one of the sequences of SEQ ID NOs: 29-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0602] Embodiment 11 Polypeptide according to embodiment 9 or embodiment 10, wherein the first ISVD has a sequence selected from SEQ ID NOs: 29-53.

[0603] Embodiment 12 Polypeptide according to any one of embodiments 9-11, wherein the second ISVD has a sequence selected from SEQ ID NOs: 29-53.

[0604] Embodiment 13 Polypeptide according to any one of embodiments 9-12, wherein the polypeptide further comprises a third ISVD capable of specifically binding to a constant domain of a human or nonhuman primate T cell receptor (TCR) present on a T cell.

[0605] Embodiment 14 Polypeptide according to embodiment 13, wherein the third ISVD has a CDR1 with amino acid sequence X1X2VHKINFX3G (SEQ ID NO: 214), a CDR2 with amino acid sequence HISIGDQTD (SEQ ID NO: 215) and a CDR3 with amino acid sequence X4SRIX5PYDY (SEQ ID NO: 216), according to Abm definition, wherein

[0606] Xi is selected from G, W, E or D;

[0607] X2 is selected from D, Y G or W;

[0608] X3is selected from L or Y;

[0609] X4is selected from F or L; and

[0610] X5is selected from W, Y, T, S or Q.

[0611] Embodiment 15 Polypeptide according to embodiment 13 or 14, wherein the third ISVD has: a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 219; or a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIYPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 259.

[0612] Embodiment 16 Polypeptide according to any one of embodiments 13-15, wherein the third ISVD has a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY); or a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIYPYDY).

[0613] Embodiment 17 Polypeptide according to any one of embodiments 13-16, wherein the third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with SEQ ID NO: 55 or SEQ ID NO: 56 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded, or wherein the third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NO: 261 to SEQ ID NO: 268 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0614] Embodiment 18 Polypeptide according to any one of embodiments 13-17, wherein the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 or SEQ ID NO: 56, or wherein the third ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NO: 261 to SEQ ID NO: 268.

[0615] Embodiment 19 Polypeptide according to any one of embodiments 13-18, wherein the third ISVD is at the N-terminal end of the polypeptide. Embodiment 20 Polypeptide according to any one of embodiments 9-19, wherein the polypeptide comprises: a. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 31, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or b. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or c. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or d. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or e. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or f. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 34, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or g. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or h. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 41 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or i. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or j. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 50, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or k. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or l. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or m. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or n. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or o. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or p. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 40, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or q. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or r. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or s. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or t. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 51, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or u. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or v. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 45, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or w. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or x. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 31, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or y. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or z. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or aa. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or bb. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or cc. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 34, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or dd. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or ee. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 41 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or ff. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or gg. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 50, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or hh. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or ii. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or jj. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or kk. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or

[0616] II. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or mm. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 40, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or nn. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or oo. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 265; or pp. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or qq. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 51, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or rr. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or ss. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 45, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268; or tt. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 268.

[0617] Embodiment 21 Polypeptide according to any one of embodiments 9-20, wherein the polypeptide further comprises a fourth ISVD that is capable of specifically binding to human serum albumin.

[0618] Embodiment 22 Polypeptide according to embodiment 21, wherein the fourth ISVD comprises i. a CDR1 that has the sequence GFTFRSFGMS (SEQ ID NO: 220) or an amino acid sequence with 2 or 1 amino acid difference with GFTFRSFGMS (according to AbM definition); ii. a CDR2 that has the sequence SISGSGSDTL (SEQ ID NO: 221) or an amino acid sequence with 2 or 1 amino acid difference with SISGSGSDTL (according to AbM definition); and ill. a CDR3 that has the sequence GGSLSR (SEQ ID NO: 222) or an amino acid sequence with 2 or 1 amino acid difference with GGSLSR (according to AbM definition).

[0619] Embodiment 23 Polypeptide according to embodiment 21 or 22, wherein the fourth ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 100- 117 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded, preferably a degree of sequence identity with SEQ ID NO: 114 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0620] Embodiment 24 Polypeptide according to any one of embodiments 21-23, wherein the fourth ISVD comprises or essentially consists of SEQ ID NO: 114.

[0621] Embodiment 25 Polypeptide according to any one of embodiments 9-24, wherein the sequence of the polypeptide comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 62-67 or 76-85 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0622] Embodiment 26 Polypeptide according to any one of embodiments 9-25, wherein the sequence of the polypeptide comprises or essentially consists of any one of SEQ ID NOs: 62-67 or 76-85.

[0623] Embodiment 27 Polypeptide according to any one of embodiments 9-26, wherein the sequence of the polypeptide comprises or essentially consists of SEQ ID NO: 82 or SEQ ID NO: 83.

[0624] Embodiment 28 Polypeptide according to any one of embodiments 9-27, wherein the sequence of the polypeptide comprises or essentially consists of SEQ ID NO: 82.

[0625] Embodiment 29 Polypeptide construct comprising

[0626] (i) a polypeptide according to any one of embodiments 9-12;

[0627] (ii) a third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell; and

[0628] (iii) an Fc region which is linked to the polypeptide via a suitable linker or hinge region. Embodiment 30 Polypeptide construct according to embodiment 29, wherein the Fc region comprises at least one or more parts, fragments, amino acid stretches or domains of the Fc portion of IgG.

[0629] Embodiment Sl Polypeptide construct according to embodiment 30, wherein the Fc region comprises or consists of an lgG4 knob-and-hole Fc with FALA mutations.

[0630] Embodiment s Polypeptide construct according to embodiment 31, wherein the third ISVD is at the N- terminal position of the hole chain of the Fc region.

[0631] Embodiment S3 Polypeptide construct according to any one of embodiments 29-32, wherein the third ISVD has a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 219; or a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 217, a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 215, and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIYPYDY) or an amino acid sequence with 2 or 1 amino acid difference(s) with SEQ ID NO: 259.

[0632] Embodiment 34 Polypeptide construct according to any one of embodiments 29-33, wherein the third ISVD has a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 219 (LSRIWPYDY); or a CDR1 (AbM definition) that is the amino acid sequence of SEQ ID NO: 217 (GYVHKINFYG), a CDR2 (AbM definition) that is the amino acid sequence of SEQ ID NO: 215 (HISIGDQTD), and a CDR3 (AbM definition) that is the amino acid sequence of SEQ ID NO: 259 (LSRIYPYDY). Embodiment 35 Polypeptide construct according to any one of embodiments 29-34, wherein the third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with SEQ ID NO: 55 or SEQ ID NO: 56 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded, or wherein the third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NO: 261 to SEQ ID NO: 268 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0633] Embodiment 36 Polypeptide construct according to any one of embodiments 29-35, wherein the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 or SEQ ID NO: 56, or wherein the third ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NO: 261 to SEQ ID NO: 268.

[0634] Embodiment s? Polypeptide construct according to any one of embodiments 29-36, wherein the first and second ISVD comprised in the polypeptide are at the N-terminal position of the knob chain of the Fc region.

[0635] Embodiment 38 Polypeptide construct according to any one of embodiments 29-37, wherein the first and second ISVD have sequences that are selected from sequences that have a degree of sequence identity with one of the sequences of SEQ ID NOs: 29-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

[0636] Embodiment 39 Polypeptide construct according to any one of embodiments 29-38, wherein the first ISVD has a sequence selected from SEQ ID NOs: 29-53.

[0637] Embodiment 40 Polypeptide construct according to any one of embodiments 29-39, wherein the second ISVD has a sequence selected from SEQ ID NOs: 29-53. Embodiment 41 Polypeptide construct according to any one of embodiments 29-40, wherein a. the first ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and b. the second ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and c. the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 and SEQ ID NO: 56, or wherein the third ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NO: 261 to SEQ ID NO.: 268.

[0638] Embodiment 42 Polypeptide construct according to any one of embodiments 29-41, wherein: a. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or b. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, the second

[0639] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or c. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second

[0640] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or d. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or e. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or f. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second

[0641] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or g. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 40, the second

[0642] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or h. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or i. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or j. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or k. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or l. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 51, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or m. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 53, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or n. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 45, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or o. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or p. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or q. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or r. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second

[0643] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or s. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second

[0644] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or t. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second

[0645] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or u. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the second

[0646] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or v. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 40, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or w. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or x. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or y. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 265; or z. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second

[0647] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or aa. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 51, the second

[0648] ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or bb. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 53, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or cc. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 45, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268; or dd. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 268

[0649] Embodiment 43 Polypeptide construct according to any one of embodiments 29-42, wherein the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NOs: 68, 86, 88-92 or 94-97.

[0650] Embodiment 44 Polypeptide construct according to any one of embodiments 29-43, wherein the sequence of the knob chain of the Fc region comprises or essentially consists of any one of SEQ ID NOs: 69-74, 87 or 93.

[0651] Embodiment 45 Polypeptide construct according to any one of embodiments 29-44, wherein a. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 69; or b. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 70; or c. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 71; or d. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 72; or e. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 73; or f. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 74; or g. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 86 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or h. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0652] 88 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or i. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0653] 89 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or j. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0654] 90 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or k. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0655] 91 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or l. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0656] 92 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or m. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0657] 94 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or n. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0658] 95 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or o. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0659] 96 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or p. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:

[0660] 97 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93.

[0661] Embodiment 46 Polypeptide construct according to any one of embodiments 29-45, wherein the sequence of the construct comprises or essentially consists of the hole chain sequence of SEQ ID NO: 92 and the knob chain sequence of SEQ ID NO: 93 or the hole chain sequence of SEQ ID NO: 97 and the knob chain sequence of SEQ ID NO: 93.

[0662] Embodiment 47 Polypeptide construct according to any one of embodiments 29-46, wherein the sequence of the construct comprises or essentially consists of the hole chain sequence of SEQ ID NO: 97 and the knob chain sequence of SEQ ID NO: 93.

[0663] Embodiment 48 ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28 or polypeptide construct according to any one of embodiments 29-47, wherein the ISVD, polypeptide or polypeptide construct has an affinity for human and / or non-human primate HER2 of between 1-600 nM, preferably between 1-500 nM, more preferably between 1-100 nM, most preferably between 1-10 nM.

[0664] Embodiment 49 ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28 or polypeptide construct according to any one of embodiments 29-47, wherein the ISVD, polypeptide or polypeptide construct specifically binds to human ErbB2 and does not specifically bind to human ErbBl, human ErbB3 or human ErbB4.

[0665] Embodiment 50 A method of producing an ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28 or polypeptide construct according to any one of embodiments 29-47, wherein the method comprises: a. expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid sequence encoding the ISVD, polypeptide or polypeptide construct; optionally followed by: b. isolating and / or purifying the ISVD, polypeptide or polypeptide construct.

[0666] Embodiment s! Nucleic acid encoding the ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28 or polypeptide construct according to any one of embodiments 29-47.

[0667] Embodiment 52Vector comprising a nucleic acid according to embodiment 51.

[0668] Embodiment 53 Non-human host or host cell transformed or transfected with nucleic acid according to embodiment 51 or the vector according to embodiment 52.

[0669] Embodiment 54 Composition comprising the ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28 or polypeptide construct according to any one of embodiments 29-47.

[0670] Embodiment 55 Composition according to embodiment 54, wherein the composition is a pharmaceutical composition.

[0671] Embodiment 56 ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28, polypeptide construct according to any one of embodiments 29-47, or composition according to embodiment 54 or 55 for use as a medicament.

[0672] Embodiment 57 ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28, polypeptide construct according to any one of embodiments 29-47, or the composition according to embodiment 54 or 55, for use in the prevention, treatment or amelioration of cancer.

[0673] Embodiment 58 ISVD, polypeptide, polypeptide construct or composition for use according to embodiment 57, wherein the cancer is a HER2 positive cancer. Embodiment 59 ISVD, polypeptide, polypeptide construct or composition for use according to embodiment 58, wherein the HER2 positive cancer is selected from gastric cancer, gastroesophageal junction adenocarcinoma or breast cancer.

[0674] Embodiment 60 Method of treating cancer, wherein said method comprises administering, to a subject in need thereof, a pharmaceutically active amount of the ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28, polypeptide construct according to any one of embodiments 29-47, or the composition according to embodiment 54 or 55.

[0675] Embodiment 61 Method according to embodiment 60, wherein the cancer is a HER2 positive cancer.

[0676] Embodiment 62 Method according to embodiment 61, wherein the HER2 positive cancer is selected from gastric cancer, gastroesophageal junction adenocarcinoma or breast cancer.

[0677] Embodiment 63 Use of the ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28, polypeptide construct according to any one of embodiments 29-47, or the composition according to embodiment 54 or 55 in the preparation of a medicament.

[0678] Embodiment 64 Use of the ISVD according to any one of embodiments 1-7, polypeptide according to any one of embodiments 8-28, polypeptide construct according to any one of embodiments 29-47, or the composition according to embodiment 54 or 55 in the preparation of a medicament for treatment of cancer.

[0679] EXAMPLES

[0680] Example 1: Characterization of HER2 binding immunoglobulin single variable domains

[0681] HER2 binding immunoglobulin single variable domains (ISVDs) labelled with codes 27A05, 27E07, 29E09 and 47D05, generated as described in WO 2009 / 068625A2, were used in this Example. An ISVD binding to cAbLys3, labelled IRR00022 (SEQ ID NO.: 54), was used as negative control.

[0682] Table 1: List of HER2 binding ISVD and negative control

[0683] Affinity determination of ISVDs to human and cynomolgus monkey HER2

[0684] Affinity determinations for 27A05, 29E09 and 47D05 were done using surface plasmon resonance (SPR) on a ProteOn XPR36 instrument (BioRad Laboratories, Inc.). Human, cynomolgus monkey (cyno) and mouse HER-2 Fc proteins (GenBank NP_004439.2 Met 1- Thr 652, GenBank XP_001090430.1,Aspl21Asn,Leul22Pro Metl-Thr652 and Uniprot P70424 Metl-Thr 653, respectively, i.e., SEQ ID NO.: 269, 270 and 271, respectively) were immobilized on a ProteOn GLC Sensor Chip (20pg / mL, lOmM Acetate pH4.0, 120s, 30pL / min). Purified ISVDs were injected at six different concentrations (0.73 to 750nM) for 120 seconds and dissociation was followed for 600s-1800 seconds. Data was double referenced by subtracting a reference ligand lane and a blank buffer injection. Processed sensorgrams were analysed based on a 1:1 interaction model (Langmuir binding model) using the ProteOn Manager (Version 3.1.0.6) software.

[0685] Results of the affinity determinations can be seen in Table 2.

[0686] Table 2: SPR based affinity determination of HER2 binding ISVDs for human and cyno HER2 protein

[0687] All ISVDs had high affinity, in the nanomolar or lower range, except 27E07, for which measurements were not accurate. For this reason, steady-state affinity determinations were done for 27E07, using SPR on a Biacore 8K+ instrument (Cytiva). Both cyno and human HER-2 Fc protein were immobilized on a Biacore CM5 Chip (lOpg / mL, lOmM Acetate pH4.0, 100s, lOpL / min). Purified ISVDs were injected at 7 different concentrations (ranging from 2 uM to 15 nM) for 260 seconds and dissociation was followed for 600s-1800 seconds. This was done in multi -cycle kinetics (association from lowest to highest concentration with both dissociation and regeneration for each concentration). Data was double referenced by subtracting a reference ligand lane and a blank buffer injection. Processed sensorgrams were analysed based on a 1:1 interaction model (Langmuir binding model) using the Biacore Insight Evaluation Software (version 3.0.12).

[0688] Steady state affinity determination resulted only in an indicative value of 50 nM for human HER2 and no value for cyno HER2. However, binding to cyno HER2 was observed in the sensorgrams.

[0689] No binding to mouse HER2 was detected for any of the ISVDs.

[0690] Binding to cell-expressed HER2 in JIMT-1 and SKBR-3 cells

[0691] Binding of ISVDs to cell-expressed HER2 was confirmed on JIMT-1 (DMSZ, catalogue # ACC 589) and SKBR- 3 (ATCC, catalogue # HTB-30) cells. These cells express an estimated 1E05 and 1E06 HER2 receptors per cell, respectively.

[0692] Binding of ISVDs to cell-expressed HER2 was examined with flow cytometry. Cells were thawed, washed with assay buffer (PBS, 2% FBS, 0.05% NaN3) and seeded separately in 384-well Bio-One V-bottom plates (Greiner, Cat. No. 781280), with a total of 2.5E04 cells seeded per well. After washing, serial dilutions of ISVDs (starting from 1 pM, 4-fold dilution, 11 points, diluted in assay buffer), were added and incubated for 60 to 120 minutes at 4 °C. Plates were then washed 3 times and cells were incubated in APC labelled anti-DYKDDDDK Tag (Biolegend, #6373071637308) (0.2 pg / mL diluted in assay buffer) for 30 minutes at 4°C. Plates were finally washed 3 times and cells were resuspended in DAPI solution (BD Biosciences, Cat. No. 564907, 1000-fold diluted in assay buffer). Cell suspensions were analyzed with iQue Screener PLUS (Intellicyt). In each washing step, plates were centrifuged at 300 x g for 2 minutes at 4°C, supernatants were discarded and buffer dispensed by a microplate washer (BioTek). Other reagents were added manually. EC5o values were estimated by dose response modelling. Curves were fit using 4 parameter logistic regression in GraphPad Prism 8 (GraphPad Software Inc.).

[0693] Binding was observed on both cell lines, for all four ISVDs (Figure 1). The lower affinity to human HER2 protein of 27E07 was reflected in the low cell binding curves. However, the binding is specific since it differentiates from the negative control (IRR00022).

[0694] Epitope binning of HER2 binding ISVDs

[0695] In-tandem epitope binning to map binding sites of the 4 ISVDs against each other, Trastuzumab and Pertuzumab (in-house produced) was performed using Bio-Layer Interferometry (BLI) on an Octet RED384 instrument (Pall ForteBio Corp.). In-house biotinylated anti-human IgG Fc ISVD was immobilized on streptavidin sensors (SA, ForteBio). Sensors were first dipped into running buffer (lx HBS-EP+, pH7.4) to determine baseline setting. Subsequently, sensors were dipped into wells containing the purified ISVDs, positive controls, or benchmark for the first association step (180 seconds), followed by wells containing a panel of different purified ISVDs, positive controls, or benchmark. ISVDs pairs were assessed for competitive binding. Additional binding by the second ISVD was determined with a report point at 20 seconds of association and by visual inspection. Additional binding indicates an unoccupied epitope: noncompetitor, while no binding indicates epitope blocking: competitor.

[0696] It was determined that the 4 ISVDs belong to 3 different bins. 47D05 competed with Pertuzumab, forming one bin. The second bin consisted of 27A05 and 29E09 and the third bin consisted of 27E07. None of the clones competed with Trastuzumab.

[0697] Paralogue binding

[0698] Binding of the ISVDs to human ErbBl, human ErbB3 and human ErbB4 was examined by ELISA. 384-well high binding SpectraPlates (PerkinElmer, Cat. No. 6007509) were coated overnight at 4°C with 1 pg / mL of human ErbBl (Sino Biological, 10001-H02H), human ErbB2 (R&D Systems, 1129-ER), human ErbB3 (Sino Biological, 10201-H02H), human ErbB4 (Sino Biological, 10363-H02H) and human IgGl Isotype Control Recombinant Antibody (BioLegend, 403501) in PBS. Plates were washed (PBS, 0.05% Tween 20) and then blocked for 1 hour at RT with 1% casein (in PBS). After washing, serial dilutions of ISVDs (starting from 1 pM, 4-fold dilution, 11 points, diluted in ELISA buffer), were added to the plates and incubated for 1 hour at RT. Unbound material was washed away and bound ISVD was detected using mouse anti-FLAG-HRP (Sigma, Cat. No. A8592) (2500-fold diluted in ELISA buffer) for 1 hour at RT. After washing, es(HS)TMB was added (SDT, Cat. No. esTMB). Substrate conversion was stopped after 10 minutes by addition of 1 M HCI. Absorbance at 450 nm was measured (background absorbance measured at 620 nm was subtracted) using the CLARIOstar microplate reader (BMG Labtech).

[0699] No binding was observed to human ErbBl, human ErbB3 and human ErbB4 for any of the ISVDs, confirming specificity of the ISVDs for HER2 within the HER family members.

[0700] Biophysical properties

[0701] A thermal shift assay (TSA) was performed in a 96-well plate on the LightCycler 48011 machine (Roche). Per row, one ISVD was analyzed at one of the following pH levels: 4, 5, 6, 7, 8 and 9. Per well, 5 pL of ISVD sample (0.8 mg / mL in PBS) was added to 5 pL of Sypro Orange (40x in Mil HQ water; Invitrogen cat. No. S6551) and 10 pL of buffer (100 mM phosphate, 100 mM borate, 100 mM citrate and 115 mM NaCI, pH range 3.5 to 9). The applied temperature gradient (37 to 99°C at a rate of 0.03°C / s) induces unfolding of the ISVDs, whereby hydrophobic patches become exposed. Sypro Orange binds to those hydrophobic patches, resulting in an increase in fluorescence intensity (Ex / Em = 465 / 580 nm). The inflection point of the first derivative of the fluorescence intensity curve at pH 7 serves as a measure of the melting temperature.

[0702] The temperature at which an ISVD protein starts to aggregate (= temperature of onset of aggregation = Tagg) was determined by Dynamic Light Scattering (DLS) using the DynaPro Plate reader (Wyatt). For this, ISVDs with a 3xFLAG-HIS6 tag, produced in E. coli, purified via IMAC followed by preparative SEC, filtered (0.22 pm), at a concentration of 1 mg / mL (D-PBS) were used. After thawing, the sample was filtered over a 0.1 pm membrane and centrifuged for 5 min at 14000 rpm. Samples of 30 pL (4 replicates) were heated from 40 to 80°C at a constant rate of 0.25°C / minute with continuous recording of the light scattering intensities. The Hydrodynamic radius derived from the measured intensities was plotted against the temperature to determine the temperature at which the radius started to increase (= Tagg, °C). Oligomerization propensity of monovalent ISVDs under stressed conditions (1 week at 45°C) was investigated by analytical size exclusion chromatography (SE-HPLC). For this, ISVDs with a 3xFLAG-HIS6 tag, produced in E. coli, purified via IMAC followed by preparative SEC, filtered (0.22 pm), at a concentration of 1 mg / mL (DPBS) were used. The SE-HPLC profiles of two 100 pL aliquots were compared: one sample was incubated for 1 week at -20°C and the other sample for 1 week at 45°C. Samples were cleared by centrifugation for 5 minutes at 20000 RCF and subsequently analyzed on an Acquity UPLC BEH200 SEC (mobile phase 750 mM L-Arginine. HCI + 10 mM Phosphate pH 7.0, flowrate 0.4 mL / minute). The difference in relative pre-peak areas of stressed (+45°C) and non-stressed samples (-20°C) was calculated and reported as A% oligo (= % oligo 1W 45°C - % oligo TO).

[0703] Results are summarized in Table 3.

[0704] Table 3: Biophysical properties (Tm, Tagg and Oligomerization) of HER2 binding ISVDs

[0705] Cytotoxicity of ISVDs formatted as T cell engagers (TCE-HER2-ALB)

[0706] After showing binding of the clones to cell-expressed HER2, the HER2 binding ISVDs were formatted as multispecific T cell engaging ISVD constructs to validate the capacity to form a functional immunological synapse. The HER2 binding ISVD (HER2) was flanked by the T cell engager building block (TCE (TCE01, SEQ ID NO.: 55)) placed at the N-terminal position and the ISVD building block directed against human serum albumin (ALB) at the C-terminal position, and all linked by 9GS linkers, forming TCE-HER2-ALB ISVDs constructs. The generated constructs are listed in Table 4. The reference construct T017000698 comprised the TCEOl building block and the ALB building block linked by 9GS linker (without the HER2 binding ISVD).

[0707] Table 4: List of TCE-HER2-ALB formatted T cell engagers

[0708] Cytotoxicity of TCE-HER2-ALB T cell engagers against MCF-7 cells (ATCC HTB-22) and HCC1954 cells (ATCC catalogue # CRL-2338) was checked using the xCELLigence T cell dependent cell cytotoxicity (TDCC) assay which was performed in the culture media of the respective target cell lines and was supplemented with lx Penicillin / streptomycin (Life Technologies, cat. no.15140-122) (from now on referred to as 'assay medium'). At day 0, 50 pl assay medium was added to a 96 well E-plate (Agilent, cat. no. 5232368001). 200 pl assay medium was added to all unused wells. Plates were placed in the xCELLigence RTCA device (Agilent) and a background measurement was performed. Subsequently, the appropriate amount of target cells was seeded in the 96 well E-plate. Plates were placed back in the xCELLigence RTCA device and impedance was measured every 15 min for 24h. At day 1, 8x-concentrated compound dilution series and 8x-concentrated (240 pM) Alburex 20 human serum albumin (HSA) (CSL Behring, cat. no. 2160-679) were prepared in assay medium. 25 pl HSA solution was added to each well of the 96 well E-plate. Thereafter, 25 pl diluted compounds were added to the appropriate wells. Human T-cells (isolated previously from buffy coat) were thawed and counted. Cell suspension concentration was adjusted to yield an effectortarget (E:T) ratio of 8:1. 50 pl T-cell suspension was then added to the 96 well E-plate, resulting in a final assay volume of 200 pl. The E-plate was placed back in the xCELLigence RTCA device. Impedance was measured every 15 min for as long as desired (typically 4-5 days). For the analysis, the cell indexes of the selected timepoint were normalized to the cell index right before compound addition to account for well-to-well seeding variation.

[0709] MCF-7 and HCC1954 cells express an estimated 2E04 and 1E06 HER2 receptors per cell, respectively. Despite the big difference in HER2 expression levels, all four TCE-HER2-ALB formatted T cell engagers showed cytotoxicity against MCF-7 and HCC1954 cells (Figure 2). TCE01-27E07-ALB, being the weaker HER2 binder, also showed killing effect but with a much lower IC50 than the others formatted T cell engagers (Table 5).

[0710] Table 5: IC50 (M) of TCE-HER2-ALB formatted T cell engagers in the impedance-based human TDCC assay

[0711]

[0712] Example 2: HER2 binding ISVD epitope determination by CryoEM

[0713] In order to have a better understanding of the paratope / epitope interactions, cryogenic electron microscopy was performed on complexes of ISVDs bound to HER2.

[0714] Complex formation

[0715] Extracellular HER2 (uniport code:P04626 residues 23 to 652, see also SEQ ID NO.: 272) was purchased from Selvita. Anti-ISVD Fabs (TPP-37990, TPP-37995 and TPP-37996) were produced in-house. Pertuzumab and Trastuzumab Fabs were produced from the in-house available corresponding Mabs by papaine digestion.

[0716] To increase the size of HER2-ISVD complexes anti-ISVD Fabs where included. Four complexes were built: HER2-47D05-Fab37996, HER2-27A05-47D05-Fab37996, Her-29E09-Fab37990 and HER2-27E07-Fab37996 (to determine the epitope of 47D05, 27A05, 29E09 and 27E07 respectively).

[0717] For each complex, 1 nmol of HER2 was incubated with ISVDs at room temperature for one hour in BufferA (50mM Hepes pH7.5-150 mM NaCI) at a ratio 1:1.2. The Fab was then added to the resulting HER2-ISVD complex at a ratio complex:Fab of 1:1.2 (TPP-37995, TPP-37990 or TPP-37996 Fabs according to need). After incubation for 1 hour at room temperature the complexes were subjected to gel filtration using a Superdex200-increase-3.2 / 300 and purified fractions were used for grid preparation.

[0718] Grid preparation

[0719] For each complex 3 mL of freshly prepared gel filtration fraction was applied to glow discharged UltrAufoi I R 0.6 / 1 grids. Grid vitrification was performed in a Vitrobot apparatus (Thermo Fisher Scientific) with 95% humidity, ashless filter paper (Standard Vitrobot Filter Paper, 055 / 20 mm, Grade 595; Electron Microscopy Sciences), and using a blot time of 5 seconds while blotting force was set to 5. Grids were stored in liquid nitrogen and collected in a 200 keV Thermo Fisher Scientific Glacios Cryo-Transmission Electron Microscope. Images were recorded using a Falcon IV direct electron detector camera in linear mode. The images were collected using EPU software (Thermo Fisher Scientific), with a defocus range from -0.8 to -2.2 pm. The pixel size was set to 0.58 angstrom (A) and an overall dose of 67 e- / A2 was used. The number of micrographs collected was 13.307, 20.015, 6.063 and 9.598 for the complexes HER2-47D05-Fab37996, HER2-27A05-47D05-Fab37996, HER2-29E09-Fab37990 and HER2-27E07-Fab37996 respectively.

[0720] CryoEM data processing and model building

[0721] Movie frames alignment and CTF (Contrast Transfer Function) estimation were performed within cryoSPARC v3.2. Micrographs were quality curated and CTF-fit cut to 5. After a round of blob picking and several rounds of 2D-classification templates were created to repick. Particles were cleaned by using several rounds of ab-initio classification. Finally uniform 3D-refinement followed by non-uniformed 3D- refinement were used to obtain the desired map.

[0722] The resolution of the final reconstructions was estimated using the value at which the FSC curve fell below 0.143 at 2.8 A, 2.66 A, 3.01 A and 4.85 A for the complexes H ER2-47D05-Fab37996, HER2-27A05-47D05- Fab37996, Her-29E09-Fab37990 and HER2-27E07-Fab37996 respectively.

[0723] A structure model for HER2-PertuzumabFab-TrastuzumabFab from pdb database with code 6oge was used as a starting model for building. Structure refinement and model building were performed using Phenix vl.19.2-4158 (Python-based Hierarchical Environment for Integrated Xtallography) and COOT vO.9.6 (Crystallographic Object-Oriented Toolkit).

[0724] For the epitope determination, the amino acid residues of HER2 having atoms within 4 or 5 angstrom distance from each of the ISVDs were identified and are listed in Table 6. Finally, Figure 3 shows these residues in the context of the 3D structure of HER2.

[0725] Table 6: Residues within HER2 at a distance of 4 or 5 A from the ISVD as determined by Cryo-EM.

[0726] Corroborating with the results of the epitope binning described in Example 1, 27A05 and 29E09 have overlapping epitopes while 27E07 and 47D05 have distinct epitopes (Figure 4.A). Moreover, all the ISVDs bind to epitopes distal from the trastuzumab epitope and 47D05 is the only ISVD that has an overlapping epitope with pertuzumab (Figure 4.B).

[0727] Example 3: Generation and characterization of T cell engaging bivalent HER2 binding ISVD constructs in ISVD-only and ISVD-Fc formats

[0728] Generation of bivalent HER2 binding T cell engagers in ISVD-only and ISVD-Fc formats

[0729] Further multispecific T cell engaging ISVD constructs were generated using bivalent HER2 binding ISVDs, in order to screen for constructs that can distinguish, in the TDCC assay, between human tumor cell lines expressing low levels of HER2 (HER2low) and normal human cells expressing normal HER2 levels (HER2normal). ZR-75-1 cells (ATCC CRL-1500), expressing around 6E04 HER2 receptors per cell, were used as a model for HER2low and human cardiac myocytes (PromoCell GmbH) were used as a model for HER2normal, expressing about 6E03 HER2 receptors per cell. Two different formats were tested:

[0730] 1) ISVD-only format consisted of T cell engager building block (TCE01), placed at the N-terminal position, followed by two of the same or two different HER2 binding ISVDs and the ISVD building block directed against human serum albumin (ALB) at the C-terminal position (schematically exemplified in Figure 5A).

[0731] 2) ISVD-Fc format consisted of T cell engager building block (TCE01), placed at the N-terminal position of the hole chain and two of the same or different HER2 binding ISVDs placed at the N-terminal position of the knob chain (schematically exemplified in Figure 5B).

[0732] The generated constructs are listed in Table 7.

[0733] Table 7: List of bivalent binding T cell engaging HER2 ISVD constructs

[0734] Cytotoxicity of bivalent T cell engaging HER2 ISVD constructs in ISVD-only and ISVD-Fc formats Cytotoxicity of the bivalent HER2 binding ISVD constructs on ZR-75-1 cells and human cardiac myocytes (HCM) was checked using the xCELLigence TDCC assay which was performed as described in Example 1 here above.

[0735] The results are shown in Table 8. All constructs showed differentiation in killing potency between ZR-75-1 cells (HER2low) and HCM (H ER2normal) from 7.5-fold to more than 100-fold.

[0736] Table 8: IC50 (M) of HER2 binding ISVD constructs in the impedance-based human T cell mediated ZR- 75-1 cells and human cardiac myocytes cell TDCC assay

[0737]

[0738] Looking at the dose response curves (DRC), it was also observed that a significant difference in efficacy (total killing vs partial killing) was present. As shown in Figure 6, xCelligence DRC indicate a clear lower efficacy on HCM (partial killing, Figure 6B) compared to ZR-75-1 cells (total killing, Figure 6A).

[0739] Induction of proliferation of ZR-75-1 cells by bivalent HER2 binding ISVD constructs. Given the bivalency for HER2 binding, the constructs were tested in a proliferation assay. High local concentrations of HER2 can cause receptor dimerization, hence the hypothesis that engaging two HER2 receptors with the same molecule, may lead to homodimerization and unwanted signaling.

[0740] At the start of the assay lOOpl / well of ZR-75-1 cells cell suspension was seeded at 7000cells / well in 96- well clear flat bottom polystyrene TC-treated microplates (Corning, cat. no.3596). Cell suspension was prepared in RPMI 1640 medium (Life Technologies, cat. no.72400) supplemented with 10% heat- inactivated FBS (Sigma-Amdrich, cat. no.F7524), ImM sodium pyruvate (Life Technologies, cat. no.11360), 1% penicillin-strepotomycin (Life Technologies, cat. no.15140) and 30pM HSA (Alburex, CSL Behring, cat. no.2160-679). Edge wells were filled with 200pL assay medium and were not used any further in the assay. After 15min of resting at room temperature the plates were incubated in the IncuCyte (Sartorius) which stands in an incubator (37°C, 5% CO2, high humidity). 24h after seeding, the medium was removed. For starvation of the cells lOOpl / well of medium with all previously mentioned supplements except FBS was added. After lOmin of resting at room temperature the plates were placed back in the IncuCyte. Serially diluted ISVD constructs were prepared in medium (with all aforementioned supplements but 20% instead of 10% FSB and 60pM HSA instead of 30pM; final plate concentration 10% FBS and 45pM HSA). After 24h of starvation lOOpL / well of the tested compounds were added to the plates. 6 points of 1 / 10 serially diluted curves were applied, starting at lOOnM. As a positive control human EGF (R&D Systems, cat. no.236-EG-200) was used, 6 points of a 1 / 10 serially diluted curve was used, starting at lOOnM. In each plate at least 6 zero-dose (OnM compound) wells were included. The plates were sealed with Breathe Easy stickers (Diversified Biotech, cat. no.BEM-1) and after lOmin of resting at room temperature the plates were placed back in the IncuCyte until the time of analysis.

[0741] The results are shown in Figure 7. Similar to EGF, the positive control, A032800034 and A032800068, as ISVD-only formats, and A0328-00_TP020, TP-021 and TP-028, as ISVD-Fc formats, induce unwanted ZR-75- 1 proliferation. The other tested constructs showed a similar behavior as the negative controls, T017000698 and A-0328-00_TP-041, meaning no, or limited, unwanted ZR-75-1 proliferation was observed. Despite the bivalent nature of all the molecules and consequent risk of HER2 receptor homodimerization, some of the molecules in both formats did not induce ZR-75-1 proliferation, de-risking induction of tumor growth instead of tumor shrinking. Example 4: Sequence optimization and affinity dematuration of HER2 binding ISVDs

[0742] The ISVDs 27A05, 27E07, 29E09 and 47D05 were sequence optimized. Sequence optimization involves replacing one or more specific amino acid residues in the sequence in order to improve one or more (desired) properties of the ISVDs. Some examples of such sequence optimization are mentioned in the description here above.

[0743] Sequence optimization of 27A05 yielded the final sequence optimized variant A032800154, which comprises 8 amino acid substitutions L11V, A14P, V23A, P40A, N43K, D46E, V69I, E83R (Table 9) compared to the parental ISVD clone 27A05.

[0744] Sequence optimization of 27E07 yielded the final sequence optimized variant A032801441, which comprises 7 amino acid substitutions L11V, A14P, F40A, P62S, D82bS, K83R, V89L (Table 9) compared to the parental ISVD clone 27E07.

[0745] Sequence optimization of 29E09 yielded the final sequence optimized variant A032801483, which comprises 9 amino acid substitutions L11V, V23A, M34I, N43K, D46E, L48V, V69I, K83R, V89L (Table 9) compared to the parental ISVD clone 29E09.

[0746] Sequence optimization of 47D05 yielded the final sequence optimized variant A032801467, which comprises 5 amino acid substitutions L11V, S59Y, K83R, V89L, G100E (Table 9) compared to the parental ISVD clone 47D05.

[0747] The ISVDs 27A05, 29E09 and 47D05 sequences were further modified (Table 9) in order to have a high (< 10 nM), a medium (10-100 nM) and a low (>100 nM) affinity variant for each, as determined by SPR and described in Example 1 (Table 10).

[0748] Affinity dematuration of 27A05 yielded variant A032801524, with an affinity of 88 nM, and variant A032801529, with an affinity of 280 nM, which comprise 1 amino acid substitution ISIS and V58Y, respectively, compared to the ISVD optimised ISVD clone A032800154, with an affinity of 1 nM (Table 9 and Table 10). Affinity dematuration of 29E09 yielded variant A032801484, with an affinity of 25 nM, and variant A032801579, with an affinity of 500 nM, which comprise 1 amino acid substitution M34L and R32A, respectively, compared to the optimised ISVD clone A032801483, with an affinity of 13 nM (Table 9 and Table 10).

[0749] Affinity dematuration of 47D05 yielded variant A032801497, with an affinity of 110 nM, and variant A032801499, with an affinity of 360 nM, which comprise 1 amino acid substitution V72D and D76N, respectively, compared to the optimised ISVD clone A032801467, with an affinity of 6 nM (Table 9 and Table 10).

[0750] Thermal stability of the variants was tested in a thermal shift assay (TSA) using a Lightcycler (Roche). In this assay, the parental ISVDs and their variants were incubated at different pH in the presence of SYPROTM orange and a temperature gradient was applied. When an ISVD starts denaturing, SYPROTM orange binds leading to an increase in fluorescence, allowing determination of the melting temperature (Tm) for a certain pH. The results are shown in Table 10. All variants show acceptable Tm values.

[0751] Table 10: SPR-based affinity for binding human HER2 and TSA-based melting temperature of the sequence optimized and affinity dematured ISVD variants

[0752] Example 5: Generation and characterization of bivalent sequence optimized HER2 binding

[0753] ISVD constructs in ISVD-only and ISVD-Fc formats

[0754] Sequence optimized and selected affinity dematured HER2 binding ISVD variants were used for the generation of HER2 binding bivalent T cell engagers both in ISVD-only and ISVD-Fc formats (Table 11).

[0755] Table 11: List of bivalent sequence optimized HER2 binding ISVD constructs

[0756] >

[0757] Cytotoxicity of the sequence optimized bivalent HER2 binding ISVD constructs on ZR-75-1 cells and human cardiac myocytes (HCM) was checked using the xCELLigence TDCC assay which was performed as described in Example 1.

[0758] The results are shown in Figure 8. All constructs showed almost complete differentiation in killing potency and efficacy between ZR-75-1 cells (HER2low) and HCM (HER2normal).

[0759] Example 6: De-risking of T cell epitopes for the sequence optimized HER2 binding ISVD constructs

[0760] In-silico methods were used to predict potential T cell epitopes in the sequence optimized ISVD variants that comprise the HER2 binding bivalent T cell engagers in both ISVD-only and ISVD-Fc formats.

[0761] In silico antigenicity assessment of ISVD sequences was performed by the Sanofi in house Metapredictor tool. This tool uses the NetMHCIIpan-v4.0 software (http: / / www.cbs.dtu.dk / services / NetMHCIIpan / ).

[0762] Uploaded ISVD sequences were segmented into consecutive linear fragments of 15 amino-acids, called 15-mers, that differ by 1 amino acid. All 15-mers were evaluated for binding on 19 HLA-DR alleles. The panel of selected HLA-alleles consisting of DRBl_01:01, DRBl_01:02, DRBl_03:01, DRBl_04:01, DRBl_04:04, DRBl_04:05, DRBl_04:07, DRBl_07:01, DRBl_09:01, DRBl_10:01, DRB1_11:O1, DRB1_11:O4, DRB1_12:O1, DRB1_13:O1, DRB1_13:O2, DRB1_14:O1, DRB1_15:O1, DRB1_15:O2 and DRB1_15:O3, covers approximately 89% of the Caucasian population and 78% of the world population. Each 15-mer / H LA-DR combination was classified as strong, weak or no binder based on its percentile rank for both the predicted eluted ligand (EL) value and the predicted IC50 value. Applied threshold settings for this classification were <2% defined as strong binder, between 2% and 10% as weak binder and > 10% as no binder. Based on this classification and taking into account the population frequency of the interacting HLA-allele, a weighted non-human only score was calculated as a parameter for ranking of different sequences according to predicted antigenicity. Within this score all HLA-binding 9-mer core sequences that are fully human are excluded as tolerization to these peptides is anticipated.

[0763] T cell epitope de-risking yielded the final sequence optimized variant A032801618, which comprises 2 amino acid substitutions L50G and Y79D (Table 12) compared to the sequence optimized A032801467.

[0764] T cell epitope de-risking yielded the final sequence optimized variant A032801621, which comprises 2 amino acid substitutions S52D and Y79D (Table 12) compared to the sequence optimized A032801467.

[0765] T cell epitope de-risking yielded the final sequence optimized variant A032801623, which comprises 1 amino acid substitution Y79D (Table 12) compared to the sequence optimized A032801467.

[0766] T cell epitope de-risking yielded the final sequence optimized variant A032801663, which comprises 2 amino acid substitutions L50G and Y79D (Table 12) compared to the sequence optimized A032801497.

[0767] T cell epitope de-risking yielded the final sequence optimized variant A032801668, which comprises 1 amino acid substitution Y79D (Table 12) compared to the sequence optimized A032801497.

[0768] T cell epitope de-risking yielded the final sequence optimized variant A032801613, which comprises 1 amino acid substitution Y79S (Table 12) compared to the sequence optimized A032800154.

[0769] T cell epitope de-risking yielded the final sequence optimized variant A032801689, which comprises 1 amino acid substitution V57R (Table 12) compared to the sequence optimized A032801524.

[0770] T cell epitope de-risking yielded the final sequence optimized variant A032801690, which comprises 1 amino acid substitution Y79D (Table 12) compared to the sequence optimized A032801524.

[0771] T cell epitope de-risking yielded the final sequence optimized variant A032801588, which comprises 1 amino acid substitutions Y79D (Table 12) compared to the sequence optimized A032801441.

[0772] T cell epitope de-risking yielded the final sequence optimized variant A032801589, which comprises 1 amino acid substitutions V95D (Table 12) compared to the sequence optimized A032801441. T cell epitope de-risking yielded the final sequence optimized variant A032801593, which comprises 2 amino acid substitutions Y79S and V95S (Table 12) compared to the sequence optimized A032801441.

[0773] Thermal stability of the variants was tested in a thermal shift assay (TSA) as described in Example 3. All variants show acceptable Tm values (Table 12).

[0774] Table 12: List of T cell epitope de-risking HER2 binding ISVD variants and respective TSA-based melting temperature values

[0775] Example 7: Generation of sequence optimized and T cell epitope de-risked HER2 binding ISVD constructs as ISVD-only and ISVD-Fc formats

[0776] New bivalent HER2 binding ISVD constructs were generated based on the constructs of Table 12 with the following differences:

[0777] 1. TCE01 was substituted by TCE03a (EID) (SEQ ID NO.: 56).

[0778] 2. T cell epitope de-risking of HER2 binding ISVD variants were introduced.

[0779] All constructs generated are listed in Table 13. All constructs listed showed an acceptable production yield. Table 13: List of bivalent sequence optimized and T cell epitope de-risked HER2 binding ISVD constructs

[0780] Cytotoxicity of bivalent HER2 binding ISVD constructs in ISVD-only formats on ZR-75-1 cells was checked using the xCELLigence TDCC assay which was performed as described in Example 1.

[0781] The results are shown in Figure 9. Of the 5 bivalent HER2 binding ISVD constructs A032801765, A032801777 and A032801785 showed the lowest potency and A032801765 and A032801785 showed loss of efficacy (partial killing). A032801773 and A032801783 remained potent molecules.

[0782] Induction of ZR-75-1 cell proliferation of bivalent HER2 binding ISVD constructs in ISVD-Fc formats was checked using the Incucyte proliferation assay, which was performed as described in Example 4.

[0783] The results are shown in Figure 10. Of the 5 bivalent HER2 binding T cell engagers in ISVD-Fc formats, TPP- 45136, TPP-45146 and TPP-45160 induce undesirable ZR-75-1 cell proliferation, similar to the positive control, EGF. TPP-45131 and TPP-45142 did not induce ZR-75-1 cell proliferation, similar to the negative control TPP-45161. As such, these two constructs were selected for further testing. Example 8: In vitro mode of action characterization of sequence optimized and T cell epitope de-risked T cell engaging bivalent HER2 binding ISVD constructs

[0784] Binding characterization of A032801773, A032801783, TPP-45131 and TPP-45142

[0785] Kinetic binding parameters and affinities for the ISVD constructs A032801773, A032801783, TPP-45131 and TPP-45142 towards human and cynomolgus monkey HER2, TCRa0 zipper protein and serum albumin were determined through surface plasmon resonance (SPR). The SPR studies were performed on a Biacore 8K+ instrument (Cytiva) or SPR-32 instrument (Bruker Daltonics) at 25°C.

[0786] For the binding of constructs TPP-45131 and TPP-45142 to HER2 and TCR, human HER2 (Sino Biological, cat# 90295-C02H), cynomolgus HER2 (Sino Biological, cat# 10004-H02H), human TCRa0 zipper protein (inhouse generated) and cynomolgus monkey TCRa0 zipper protein (inhouse generated) were immobilized in different spots of a Cl chip (Cytiva) using Cytiva's amine coupling kit according to the manufacturer's instructions. Different concentrations between 800 nM and 45 nM of constructs TPP-45131 and TPP-45142 were injected for 2 minutes and allowed to dissociate for 30 minutes at a flow rate of 30 pl / min. Between sample injections, the surfaces were regenerated with 10 mM glycine pHl,5. As running buffer HBS-P+ buffer (Cytiva) was used. The kinetic rate constants (kaand kd) were calculated by fitting the sensorgrams with the Biacore 8K+ Evaluation Software using a 1:1 Langmuir binding model with mass transport limitations. The equilibrium dissociation constant KDwas calculated by the software as the kd / karatio.

[0787] For the binding of constructs A032801773 and A032801783 to HER2 and TCR, human HER2 (Sino Biological, cat# 90295-C02H ), cynomolgus HER2 (Sino Biological, cat# 10004-H02H), human TCRa0 zipper protein (inhouse generated) and cynomolgus monkey TCRa0 zipper protein (inhouse generated) were immobilized on different spots of a high capacity amine chip (Bruker Daltonics, cat#1862614) using the Amine Coupling Kit II (Bruker Daltonics, cat#1862635) according to the manufacturer's instructions. Different concentrations between 800 nM and 2,7 nM of constructs A032801773 and A032801783 were injected for 2 minutes and allowed to dissociate for 15 minutes at a flow rate of 30 pl / min. Between sample injections, the surfaces were regenerated with 10 mM glycine pHl,5. As running buffer HBS-P+ buffer (Cytiva) was used. The kinetic rate constants (kaand kd) were calculated by fitting the sensorgrams with Sierra Analyser 3.4.1 software using a 1:1 Langmuir binding model. The equilibrium dissociation constant KDwas calculated by the software as the kd / karatio. The results for binding of both formats of constructs to HER2 and TCR are shown in Table 14.

[0788] Table 14: SPR based affinity determination of A032801773, A032801783, TPP-45131 and TPP-45142 for human and cynomolgus TCR and HER2 proteins

[0789] For the binding study on serum albumin, human serum albumin (Sigma Aldrich, cat# A8763) and cynomolgus monkey serum albumin (inhouse produced) were immobilized on a Amine Flat Sensor chip (Bruker Daltonics, cat# 1862611) using the Amine Coupling Kit II according to the manufacturer's instructions. Different concentrations between 1000 nM and 1,6 nM of constructs A032801773 and A032801783 were injected for 2 minutes and allowed to dissociate for 15 minutes at a flow rate of 45 pl / min. Between sample injections, the surfaces were regenerated with 100 mM HCI. As running buffer HBS-P+ buffer (Cytiva) was used. The kinetic rate constants (kaand kd) were calculated by fitting the sensorgrams with Sierra Analyser 3.4.1 software using a 1:1 Langmuir binding model. The equilibrium dissociation constant KDwas calculated by the software as the kd / karatio.

[0790] The results are shown in Table 15.

[0791] Table 15: SPR based affinity determination of A032801773 and A032801783 for human and cynomolgus albumin proteins To make sure that the ISVD constructs bind specifically to HER2 and not to other members of the ErbB family, A032801773, A032801783, TPP-45131 and TPP-45142 were tested for binding towards human HER1, HER3, and HER4 by biolayer interferometry (BLI) on an Octet HTX instrument (Pall ForteBio Corp.). Human HER2 (Sino Biological, cat# 90295-C02H) and the family members human HER1 (Sino Biological, cat# 10001-H02H), human HER3 (Sino Biological, cat# 10201-H02H) and human HER4 (Sino Biological, cat# 10363-H02H) were immobilized on Octet AR2G Biosensors (Pall ForteBio Corp., cat# 18- 5092) till high densities (between 2,4 and 3,3 nm). To verify the binding of the compounds, sensors were first dipped for 60 seconds into running buffer (lx HBS-EP+, pH7.4) to determine the baseline and subsequently for 300 seconds into wells containing A032801773, A032801783, TPP-45131 or TPP-45142 at a concentration of 1 pM in running buffer, or wells containing respective positive control antibody at a concentration of 100 nM. Data were analyzed with the ForteBio data analysis software.

[0792] Respective positive control antibodies showed binding to the ErbB family members and no binding of A032801773, A032801783, TPP-45131 and TPP-45142 was detected showing their specificity to HER2 within the ErbB family.

[0793] Cytotoxicity of A032801773, A032801783, TPP-45131 and TPP-45142 on cancer cell lines expressing different levels of HER2

[0794] Cytotoxicity of A032801773, A032801783, TPP-45131 and TPP-45142 was checked using the xCELLigence TDCC assay on different cancer cell lines with HER2 expression levels from 7k to 1000k HER2 receptors per cell. The assay was performed as described in Example 1 using HCC1954 (ATCC CRL-2338), ZR-75-1 (ATCC CRL-1500), BT20 (ATCC HTB-19) and BT549 (ATCC HTB-122) cells and 3 different T cell donors with an effector target ratio of 5:1 and in the presence of 30 uM HSA.

[0795] The results are summarized in Table 16. All molecules are potent towards the different cell lines, remarkably showing potency in the picomolar range in HER2low cell lines like BT20 and BT549.

[0796] Table 16: IC50 (M) of T cell engaging bivalent HER2 binding ISVD constructs in the impedance-based human T cell mediated cancer cell TDCC assay

[0797] Cytotoxicity of A032801773, A032801783, TPP-45131 and TPP-45142 on BT20 3D spheroids expressing HER2low

[0798] In order to increase the stringency of the TDCC assay, the HER2low BT20 cell line was used in a Incucyte- based T cell mediated spheroid TDCC assay with a decreased effector to target ratio of 1 to 5.

[0799] BT-20 Cytolight Green (in-house transduced) cells were subcultured in MEM, GlutaMAX™ Supplement (Life Techn. - Gibco, cat# 41090-028) supplemented with 10% Fetal Bovine Serum (Sigma, cat# F7524), 1 mM sodium pyruvate (Life Techn. - Gibco, cat# 11360-039) and 5 pg / ml puromycin (Life Techn. - Gibco, cat# A11138-03), before use in the assay. The spheroid assay was performed in the culture medium supplemented with lx Penicilin-Streptomycin (Life Techn. - Gibco, cat# 15140-163) and without puromycin (from now on referred to as assay medium). At day 0 of the assay, BT-20 Cytolight Green cells were detached with Tryple Express (Life Techn. - Gibco, cat# 12605), resuspended in assay medium and counted. The cells were seeded at a density of 2000 cells / well in 50 pl assay medium in a 384 ULA spheroid microplate (Corning, cat# 4516). The assay plate was incubated in Incucyte® at 37°C, 5% CO2for 2 days to allow spheroid formation. At day 2 of the assay, an 8x-concentrated IncuCyte™ Cytotox Red Reagent (Essen BioScience, cat# 4632) solution ...

Claims

CLAIMS1. Immunoglobulin single variable domain (ISVD) which specifically binds to human and / or non-human primate HER2, wherein said ISVD essentially consists of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively), wherein a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO:1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XSIX6R G TS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, whereinXi is selected from I and S;X2is selected from V, R and Y;X3is selected from A and R;X4is selected from I, L and M;X5is selected from G and L;X6is selected from D and S;X7is selected from E and G;Xg is selected from D, E and V; and wherein the full amino acid sequence of the ISVD is different from SEQ ID NOs: 29-32.

2. ISVD according to claim 1, wherein a. the amino acid sequence of CDR1 is SEQ ID NO: 1, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 13-16, and the amino acid sequence of CDR3 is SEQ ID NO: 3, according to AbM definition; orb. the amino acid sequence of CDR1 is selected from SEQ ID NOs: 17-20, the amino acid sequence of CDR2 is SEQ ID NO: 5, and the amino acid sequence of CDR3 is SEQ ID NO: 6, according to AbM definition; or c. the amino acid sequence of CDR1 is SEQ ID NO: 7, the amino acid sequence of CDR2 is selected from SEQ ID NOs: 21,23 and 24, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 22 and 25; according to AbM definition; or d. the amino acid sequence of CDR1 is SEQ ID NO: 10, the amino acid sequence of CDR2 is SEQ ID NO: 11, and the amino acid sequence of CDR3 is selected from SEQ ID NOs: 26-28; according to AbM definition.

3. ISVD according to claim 1 or claim 2, wherein a. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 13, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or b. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 14, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or c. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 15, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or d. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 1, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 16, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 3; according to AbM definition; or e. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 17, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; orf. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 18, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or g. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 19, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or h. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 20, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 5, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 6; according to AbM definition; or i. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or j. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or k. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 22; according to AbM definition; or l. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 21, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or m. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 23, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or197n. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 7, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 24, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 25; according to AbM definition; or o. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 26; according to AbM definition; or p. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 27; according to AbM definition; or q. the amino acid sequence of CDR1 comprises or essentially consists of SEQ ID NO: 10, the amino acid sequence of CDR2 comprises or essentially consists of SEQ ID NO: 11, and the amino acid sequence of CDR3 comprises or essentially consists of SEQ ID NO: 28; according to AbM definition.

4. ISVD according to any one of claims 1-3, wherein the ISVD is a heavy-chain ISVD.

5. ISVD according to any one of claims 1-4, wherein the ISVD is selected from a VHH, a humanized VHH, a camelized VH, a domain antibody, a single domain antibody, and a dAb.

6. ISVD according to any one of claims 1-5, wherein the ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with one of the sequences of SEQ ID NOs: 33-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

7. ISVD according to any one of claims 1-6, wherein the ISVD comprises or consists of a sequence selected from SEQ ID NOs: 33-53.

8. Polypeptide comprising an ISVD according to any one of claims 1-7.1989. Polypeptide comprising a first and second ISVD capable of specifically binding to human and / or nonhuman primate HER2, wherein said first and second ISVD essentially consist of 4 framework regions (FR1 to FR4 respectively) and 3 complementarity determining regions (CDR1 to CDR3 respectively, wherein the first and / or second ISVD has CDR sequences selected from the following: a. the amino acid sequence of CDR1 is GIPFSTRTMA (SEQ ID NO: 1), the amino acid sequence of CDR2 is TX1RSGAPX2 (SEQ ID NO: 2), and the amino acid sequence of CDR3 is VNGDI (SEQ ID NO: 3), according to AbM definition; or b. the amino acid sequence of CDR1 is RIPASIX3TX4A (SEQ ID NO: 4), the amino acid sequence of CDR2 is TIGSSGTPA (SEQ ID NO: 5), and the amino acid sequence of CDR3 is VNGDY (SEQ ID NO: 6), according to AbM definition; or c. the amino acid sequence of CDR1 is GSIFGFNDMA (SEQ ID NO: 7), the amino acid sequence of CDR2 is XSIX6RVGVTS (SEQ ID NO: 8), and the amino acid sequence of CDR3 is DQRLDX7STLAY (SEQ ID NO: 9), according to AbM definition; or d. the amino acid sequence of CDR1 is GITFRRYDMG (SEQ ID NO: 10), the amino acid sequence of CDR2 is TILSEGDTN (SEQ ID NO: 11), and the amino acid sequence of CDR3 is XgWRAIGRTY (SEQ ID NO: 12), according to AbM definition, whereinXi is selected from I and S;X2 is selected from V, R and Y;X3is selected from A and R;X4is selected from I, L and M;X5is selected from G and L;X6is selected from D and S;X7is selected from E and D;X8is selected from D, E and V; and wherein the second ISVD has a sequence that may be the same or different from the first ISVD.

10. Polypeptide according to claim 9, wherein the first and second ISVD have sequences that are selected from sequences that have a degree of sequence identity with one of the sequences of SEQ ID NOs: 29-53 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.19911. Polypeptide according to claim 9 or claim 10, wherein the first ISVD has a sequence selected from SEQ ID NOs: 29-53.

12. Polypeptide according to any one of claims 9-11, wherein the second ISVD has a sequence selected from SEQ ID NOs: 29-53.

13. Polypeptide according to any one of claims 9-12, wherein the polypeptide further comprises a third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell.

14. Polypeptide according to claim 13, wherein the third ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with SEQ ID NO: 55 or SEQ ID NO: 56 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

15. Polypeptide according to claim 13 or claim 14, wherein the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 or SEQ ID NO: 56.

16. Polypeptide according to any one of claims 13-15, wherein the third ISVD is at the N-terminal end of the polypeptide.

17. Polypeptide according to any one of claims 9-16, wherein the polypeptide comprises: a. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 31, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or b. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or200c. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or d. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 30, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or e. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 32, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 29 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or f. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 34, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or g. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or h. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 41 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or i. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or j. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 50, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or k. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or l. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 47 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or201m. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or n. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or o. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 39, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or p. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 40, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or q. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36, and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or r. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 55; or s. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or t. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 51, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 33 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or u. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 53, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or v. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 45, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56; or202w. a first ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 48, a second ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 36 and a third ISVD comprising or essentially consisting of the sequence of SEQ ID NO: 56.

18. Polypeptide according to any one of claims 9-17, wherein the polypeptide further comprises a fourth ISVD that is capable of specifically binding to human serum albumin.

19. Polypeptide according to claim 18, wherein the fourth ISVD comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 100-117 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded, preferably a degree of sequence identity with SEQ ID NO: 114 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

20. Polypeptide according to claim 19, wherein the fourth ISVD comprises or essentially consists of SEQ ID NO: 114.

21. Polypeptide according to any one of claims 9-20, wherein the sequence of the polypeptide comprises or essentially consists of a sequence that has a degree of sequence identity with any one of SEQ ID NOs: 62-67 or 76-85 of at least 85%, preferably at least 90%, more preferably at least 95%, even more preferably at least 99%, in which for the purposes of determining the degree of sequence identity, the amino acid residues that form the CDR sequences are disregarded.

22. Polypeptide according to any one of claims 9-21, wherein the sequence of the polypeptide comprises or essentially consists of any one of SEQ ID NOs: 62-67 or 76-85.

23. Polypeptide according to any one of claims 9-22, wherein the sequence of the polypeptide comprises or essentially consists of SEQ ID NO: 82 or SEQ ID NO: 83.20324. Polypeptide according to any one of claims 9-23, wherein the sequence of the polypeptide comprises or essentially consists of SEQ ID NO: 82.

25. Polypeptide construct comprising(i) a polypeptide according to any one of claims 9-12;(ii) a third ISVD capable of specifically binding to a constant domain of a human or non-human primate T cell receptor (TCR) present on a T cell; and(iii) an Fc region which is linked to the polypeptide via a suitable linker or hinge region.

26. Polypeptide construct according to claim 25, wherein the Fc region comprises at least one or more parts, fragments, amino acid stretches or domains of the Fc portion of IgG.

27. Polypeptide construct according to claim 26, wherein the Fc region comprises or consists of an lgG4 knob-and-hole Fc with FALA mutations.

28. Polypeptide construct according to claim 27, wherein the third ISVD is at the N-terminal position of the hole chain of the Fc region.

29. Polypeptide construct according to claim 27 or claim 28, wherein the first and second ISVD are at the N-terminal position of the knob chain of the Fc region.

30. Polypeptide construct according to any one of claims 25-29, wherein a. the first ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and b. the second ISVD comprises or essentially consists of a sequence selected from any one of SEQ ID NOs: 29-53; and c. the third ISVD comprises or essentially consists of a sequence selected from SEQ ID NO: 55 and SEQ ID NO: 56.

31. Polypeptide construct according to any one of claims 25-30 wherein:204a. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or b. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or c. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 29, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or d. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or e. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or f. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 39, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or g. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 40, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or h. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 31, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 32, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or i. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or j. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 55; or205k. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, the second ISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or l. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 51, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 33, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or m. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 53, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or n. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 45, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56; or o. the first ISVD comprises or essentially consists of the sequence of SEQ ID NO: 48, the secondISVD comprises or essentially consists of the sequence of SEQ ID NO: 36, and the third ISVD comprises or essentially consists of the sequence of SEQ ID NO: 56.

32. Polypeptide construct according to any one of claims 27-31, wherein the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NOs: 68, 86, 88-92 or 94-97.

33. Polypeptide construct according to any one of claims 27-32, wherein the sequence of the knob chain of the Fc region comprises or essentially consists of any one of SEQ ID NOs: 69-74, 87 or 93.

34. Polypeptide construct according to any one of claims 27-33, wherein a. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 69; or b. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 70; or c. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 71; or206d. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 72; or e. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 73; or f. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 68 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 74; or g. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 86 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or h. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:88 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or i. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:89 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or j. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:90 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or k. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:91 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 87; or l. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:92 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or m. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO: 94 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or207n. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:95 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or o. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:96 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93; or p. the sequence of the hole chain of the Fc region comprises or essentially consists of SEQ ID NO:97 and the sequence of the knob chain of the Fc region comprises or essentially consists of SEQ ID NO: 93.

35. Polypeptide construct according to any one of claims 27-34, wherein the sequence of the construct comprises or essentially consists of the hole chain sequence of SEQ ID NO: 92 and the knob chain sequence of SEQ ID NO: 93 or the hole chain sequence of SEQ ID NO: 97 and the knob chain sequence of SEQ ID NO: 93.

36. Polypeptide construct according to any one of claims 27-35, wherein the sequence of the construct comprises or essentially consists of the hole chain sequence of SEQ ID NO: 97 and the knob chain sequence of SEQ ID NO: 93.

37. ISVD according to any one of claims 1-7, polypeptide according to any one of claims 8-24 or polypeptide construct according to any one of claims 25-36, wherein the ISVD, polypeptide or polypeptide construct has an affinity for human and / or non-human primate HER2 of between 1-600 nM, preferably between 1-500 nM, more preferably between 1-100 nM, most preferably between 1- 10 nM.

38. A method of producing an ISVD according to any one of claims 1-7, polypeptide according to any one of claims 8-24 or polypeptide construct according to any one of claims 25-36, wherein the method comprises: a. expressing, in a suitable host cell or host organism or in another suitable expression system, a nucleic acid sequence encoding the ISVD, polypeptide or polypeptide construct; optionally followed by: b. isolating and / or purifying the ISVD, polypeptide or polypeptide construct.20839. Nucleic acid encoding the ISVD according to any one of claims 1-7, polypeptide according to any one of claims 8-24 or polypeptide construct according to any one of claims 25-36.

40. Vector comprising a nucleic acid according to claim 39.

41. Non-human host or host cell transformed or transfected with nucleic acid according to claim 39 or the vector according to claim 40.

42. Composition comprising the ISVD according to any one of claims 1-7, polypeptide according to any one of claims 8-24 or polypeptide construct according to any one of claims 25-36.

43. Composition according to claim 42, wherein the composition is a pharmaceutical composition.

44. ISVD according to any one of claims 1-7, polypeptide according to any one of claims 8-24, polypeptide construct according to any one of claims 25-36 or the composition according to claim 42 or 43, for use as a medicament.

45. ISVD according to any one of claims 1-7, polypeptide according to any one of claims 8-24, polypeptide construct according to any one of claims 25-36 or the composition according to claim 42 or 43, for use in the prevention, treatment or amelioration of cancer.

46. ISVD, polypeptide, polypeptide construct or composition for use according to claim 45, wherein the cancer is a HER2 positive cancer.

47. ISVD, polypeptide, polypeptide construct or composition for use according to claim 46, wherein the HER2 positive cancer is selected from gastric cancer, gastroesophageal junction adenocarcinoma or breast cancer.209

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