WO2014076177A1 - New polypeptide - Google Patents

Abstract

The present disclosure relates to a class of engineered polypeptides having a binding affinity for Human Epidermal Growth Factor Receptor 3 (HER3), and provides a HER3 binding polypeptide comprising the sequence LAX₃AKX₆X₇AX₉X₁₀ X₁₁LDX₁₄X₁₅GVSDX₂₀ YKX₂₃LIDKAKT VEGVX₃₅ALX₃₈X₃₉X₄₀ ILX₄₃ALP. The present disclosure also relates to said HER3 binding polypeptide also having an affinity for albumin. Furthermore, the present disclosure relates to the use of such a HER3 binding polypeptide as a diagnostic agent and/or a medicament.

Description

NEW POLYPEPTIDE

Field of the invention

The present disclosure relates to a class of engineered polypeptides having a binding affinity for Human Epidermal Growth Factor Receptor 3 (in the following interchangeably referred to as HER3 or ErbB3). The present disclosure also relates to said HER3 binding polypeptide having affinity for albumin. Furthermore, the present disclosure relates to use of such a HER3 binding polypeptide as a diagnostic agent and/or a medicament.

Background

HER3 and its role in cancer diseases

The epidermal growth factor family of transmembrane tyrosine kinase receptors, including EGFR (ErbB1 or HER1 ), ErbB2 (HER2), ErbB3 (HER3) and ErbB4 (HER4) are involved in regulating key cellular functions (e.g. cell proliferation, survival, differentiation and migration) through a complex network of intracellular signaling pathways. HER3 differs from the other receptors of this family due to its inactive tyrosine kinase domain, and hence signals via ligand-induced heterodimer formation with other tyrosine kinase receptors (Guy et al, Proc Natl Acad Sci 91 : 8132-8136 (1994); Sierke et al, Biochem J 322 (Pt 3): 757-763 (1997)). As a result, the implication of this receptor in tumor progression has long been a mystery. Recently, however, HER3 has gained interest as an allosteric kinase activator of its family members. Especially the heterodimer formed by HER2 and HER3 is said to be an exceptionally strong activator of downstream intracellular signaling (Jura et al, Proc Natl Acad Sci 106: 21608-21613 (2009); Citri et al, Exp Cell Res 284: 54-65 (2003)). This HER2-HER3 signaling pair has even been suggested as an oncogenic unit in HER2-driven breast cancer (Holbro et al, Proc Natl Acad Sci 100: 8933-8938 (2003)). In addition, up-regulation of the HER3 receptor has been shown to play an important role for the resistance to tyrosine kinase inhibitors in breast cancers overexpressing HER2 in vitro and in vivo (Sergina et al, Nature 445:437-441 (2007); Kong et al, PLoS One 3:e2881 (2008); Garrett et al, Proc Natl Acad Sci 108:5021 -5026 (201 1 )).

However, the importance of HER3 in human cancers is not limited to HER2-driven breast cancers. HER3 has also been shown to be required for tumorigenicity of HER3-overexpressing prostate cancer xenografts in vivo, to maintain in vivo proliferation of a subset of ovarian cancers via an autocrine signaling loop, and to be involved in endocrine resistance of ER⁺ breast cancer cell lines, to name a few examples (Soler et al, Int J Cancer 125:2565- 2575 (2009); Sheng Q et al, Cancer Cell 17:412-412 (2010); Liu et al, Int J Cancer 120:1874-1882 (2007); Frogne et al, Breast Cancer Res Treat 1 14:263-275 (2009)). Together, these findings demonstrate the potential of the HER3-signaling pathway as an important therapeutic target in human cancers. In addition, HER3 expression has a prognostic value, since high levels of receptor expression are associated with significantly shorter survival time in patients with melanoma and ovarian cancers (Tanner et al, J Clin Oncol 24(26):4317-23 (2006), Reschke et al, Clin Cancer Res 14(16):5188-97 (2008)).

A relatively large fraction of recently approved therapies directed towards the EGFR and HER2 receptors is based on monoclonal antibodies. In contrast to the well investigated EGFR and HER2 receptor members of the ErbB-family, there are relatively few reports on the use of anti-HER3 antibodies. Ullrich and co-workers have reported that anti-HER3 monoclonal antibodies inhibit HER3 mediated signaling in cell models of breast cancer (van der Horst et al, Int J Cancer 1 15(4):519-27 (2005)). However, although several successful cancer therapy studies have been reported using full- length monoclonal antibodies, this class of agents is not always optimal for targeting solid tumors (neither for diagnostic nor for therapeutic pay-load purposes). Therapeutic effect is dependent on an efficient distribution of the drug throughout the tumor, and molecular imaging depends on a high ratio between tumor uptake and surrounding normal tissue.

Since tumor penetration rate (including extravasation) is negatively associated with the size of the molecule, the relatively large antibody molecule (e.g. IgG) inherently has poor tissue distribution and penetration capacity. Moreover, for molecular imaging, the extraordinarily long in vivo half-life of antibodies results in relatively high blood signals and thereby relatively poor tumor-to-blood contrasts.

Recently, much smaller HER3-specific molecules based on the three- helical bundle scaffold of the Z protein, derived from domain B of Protein A from Staphylococcus aureus, were generated using combinatorial protein engineering (Kronqvist et al, Protein Eng Des Sel 24: 385-396 (2010);

WO201 1/056124). These Z variants, with sub-nanomolar affinities for HER3, demonstrated anti-proliferative effects through blockage of ligand-induced HER3-signalling of breast cancer cell lines in vitro (Gostring et al, PLoS One 7:e40023 (2012)). These growth-inhibitory effects were further demonstrated to be a result of competitive HER3 binding between the Z variant molecules and the ligand heregulin.

However, in vivo targeting of the HER3 receptor may be challenging, due to relatively low expression of the receptor on tumor cells. Typical expression levels of 10³ to 10⁴ receptors per cell have been reported (Aguilar et al, Oncogene 18: 6050-6062 (1999); Robinson et al, Br J Cancer 99: 1415- 1425 (2008)). In addition to a high tumor uptake, a prolonged retention in the tumor is of great importance for efficient therapeutic effects of a drug. Small targeting agents have the ability to accumulate at high levels in tumors due to high vascular permeability and rapid diffusivity into the tumor (Schmidt and Wittrup, Mol Cancer Ther 8: 2861 -2871 (2009)). However, unbound proteins of low molecular weight will be cleared rapidly from the tumor and then from the circulation via the kidneys.

Serum albumin

Serum albumin is the most abundant protein in mammalian sera (40 g/l; approximately 0.7 mM in humans), and one of its functions is to bind molecules such as lipids and bilirubin (Peters, Advances in Protein Chemistry 37:161 (1985)). The half-life of serum albumin is directly proportional to the size of the animal, where for example human serum albumin (HSA) has a half-life of 19 days and rabbit serum albumin has a half-life of about 5 days (McCurdy et al, J Lab Clin Med 143:1 15 (2004)). Human serum albumin is widely distributed throughout the body, in particular in the intestinal and blood compartments, where it is mainly involved in the maintenance of osmolarity. Structurally, albumins are single-chain proteins comprising three homologous domains and totaling 584 or 585 amino acids (Dugaiczyk et al, Proc Natl Acad Sci USA 79:71 (1982)). Albumins contain 17 disulfide bridges and a single reactive thiol, C34, but lack N-linked and O-linked carbohydrate moieties (Peters, (1985), supra; Nicholson et al, Br J Anaesth 85:599 (2000)). The lack of glycosylation simplifies recombinant expression of albumin. This property of albumin, together with the fact that its three-dimensional structure is known (He and Carter, Nature 358:209 (1992)), has made it an attractive candidate for use in recombinant fusion proteins. Such fusion proteins generally combine a therapeutic protein (which would be rapidly cleared from the body upon administration of the protein per se) and a plasma protein (which exhibits a natural slow clearance) in a single polypeptide chain (Sheffield, Curr Drug Targets Cardiovacs Haematol Disord 1 :1 (2001 )). Such fusion proteins may provide clinical benefits in requiring less frequent injection and higher levels of therapeutic protein in vivo. Fusion or association with HSA results in increased in vivo half-life of proteins Serum albumin is devoid of any enzymatic or immunological function and, thus, should not exhibit undesired side effects upon coupling to a bioactive polypeptide. Furthermore, HSA is a natural carrier involved in the endogenous transport and delivery of numerous natural as well as therapeutic molecules (Sellers and Koch-Weser, "Albumin Structure, Function and Uses", eds Rosenoer VM et al, Pergamon, Oxford, p 159 (1977)). Several strategies have been reported to either covalently couple proteins directly to serum albumins or to a peptide or protein that will allow in vivo association to serum albumins. Examples of the latter approach have been described e.g. in WO91/01743. This document describes inter alia the use of albumin binding peptides or proteins derived from streptococcal protein G for increasing the half-life of other proteins. The idea is to fuse the bacterially derived, albumin binding peptide/protein to a therapeutically interesting peptide/protein, which has been shown to have a rapid clearance in blood. The thus generated fusion protein binds to serum albumin in vivo, and benefits from its longer half-life, which increases the net half-life of the fused therapeutically interesting peptide/protein.

Association with HSA results in decreased immunogenicity

In addition to the effect on the in vivo half-life of a biologically active protein, it has been proposed that the non-covalent association with albumin of a fusion between a biologically active protein and an albumin binding protein acts to reduce the immune response to the biologically active protein. Thus, in WO2005/097202, there is described the use of this principle to reduce or eliminate the immune response to a biologically active protein.

Albumin binding domains of bacterial receptor proteins

Streptococcal protein G is a bi-functional receptor present on the surface of certain strains of Streptococci and capable of binding to both IgG and serum albumin (Bjorck et al, Mol Immunol 24:1 1 13 (1987)). The structure is highly repetitive with several structurally and functionally different domains (Guss et al, EMBO J 5:1567, (1986)), more precisely three Ig-binding motifs and three serum albumin binding domains (Olsson et al, Eur J Biochem 168:319 (1987)). The structure of one of the three serum albumin binding domains has been determined, showing a three-helix bundle domain (Kraulis et al, FEBS Lett 378:190 (1996)). This motif was named ABD (albumin binding c/omain) and is 46 amino acid residues in size. In the literature, it has subsequently also been designated G148-GA3.

Other bacterial albumin binding proteins than protein G from

Streptococcus have also been identified, which contain domains similar to the albumin binding three-helix domains of protein G. Examples of such proteins are the PAB, PPL, MAG and ZAG proteins. Studies of structure and function of such albumin binding proteins have been carried out and reported e.g. by Johansson and co-workers (Johansson et al, J Mol Biol 266:859-865 (1997); Johansson et al, J Biol Chem 277:81 14-8120( 2002)), who introduced the designation "GA module" (protein G-related albumin binding module) for the three-helix protein domain responsible for albumin binding. Furthermore, Rozak et al, have reported on the creation of artificial variants of the GA module, which were selected and studied with regard to different species specificity and stability (Rozak et al, Biochemistry 45:3263-3271 ( 2006); He et al, Protein Science 16:1490-1494 (2007)). Recently, variants of the G148- GA3 domain have been developed, with various optimized characteristics. Such variants are for example disclosed in PCT publications WO00/23580 (alkali-stabilized variant denoted "ABDmut"; sequence disclosed in Figure 3 of WO00/23580), WO2009/016043 and WO2012/004384. In short, drawbacks of current antibody based cancer therapies, especially in solid tumor targeting applications, include the large size of antibodies which impairs their vascular permeability and tumor penetration capability. On the other hand, known smaller binding molecules suffer the drawback of rapid renal clearance and therefore short in vivo half-life. Thus, therapies based on smaller binding molecules have to be administered more frequently and/or in higher dose leading to stressful treatment regimes and even undesirable side effects, such as pulmonary toxicity and cardiotoxicity.

As evident from the different sections of this background description, the provision of polypeptide molecules of small size and with a high affinity for HER3 as well as increased in vivo half-life could be desirable for the development of efficient therapies targeting various forms of cancer, for example breast cancer, ovarian cancer and prostate cancer. Disclosure of the invention

It is an object of the present invention to provide a molecule allowing for efficient therapy targeting various forms of cancer and other HER3 related conditions while alleviating the abovementioned and other drawbacks of current therapies.

It is another object of the present disclosure to provide a HER3 binding polypeptide, which may allow for better tissue penetration than existing molecules, while simultaneously exhibiting an increased in vivo half-life.

These and other objects which are evident to the skilled person from the present disclosure are met by different aspects of the invention as claimed in the appended claims and as generally disclosed herein.

Thus, in the first aspect of the disclosure, there is provided a HER3 binding polypeptide, comprising an amino acid sequence selected from: i) LAX3AKX6X7AX9X10 Xi i LDXi₄Xi₅GVSDX₂o YKX₂₃LIDKAKT

VEGVX35ALX38X39X40 ILX43ALP wherein, independently of each other,

X3 is selected from D, Q, R, S and T;

Xe is selected from A, K, R and T;

X₇ is selected from L, R and V;

X9 is selected from L and N;

X10 is selected from H, R and Y;

X11 is selected from F, I, L, M and V;

Xi4 is selected from A, D, E, G, H, K, L, M, N, P, Q, R, S, T and V;

Xi₅ is selected from K, R, T and V;

X20 is selected from F and Y;

X23 is selected from D and R;

X35 is selected from H, M, Q and R;

X38 is selected from A, I, L, S, T and V;

X39 is selected from F, I, L, R and S;

X40 is selected from A and E; and

X₄₃ is selected from A, G, H, I, L, P, R, T and V; and ii) an amino acid sequence which has at least 93 % identity to the

sequence defined in i).

As the skilled person will realize, the function of any polypeptide, such as the HER3 binding capacity of the polypeptides of the present disclosure, is dependent on the tertiary structure of the polypeptide. It may therefore be possible to make minor changes to the sequence of amino acids in a polypeptide without affecting the function thereof. Thus, the invention encompasses modified variants of the HER3 binding polypeptide, which are such that the HER3 binding characteristics are retained. In another

embodiment the invention encompasses modified variants of the HER3 binding polypeptide which retain HER3 binding characteristics and albumin binding characteristics.

In this way, also encompassed by the present disclosure is a HER3 binding polypeptide comprising an amino acid sequence with 93 % or greater identity to a polypeptide as defined in i). In some embodiments, the inventive polypeptide may comprise a sequence which is at least 95 %, such as at least 97 %, identical to the polypeptides as defined in i).

In some embodiments, such changes may be made in all positions of the sequences of the HER3 binding polypeptide as disclosed herein. In other embodiments, such changes may be made only in the non-variable positions, also denoted "scaffold" amino acid residues. In such cases, changes are not allowed in the variable positions, i.e. positions denoted with an "X" in sequence i). When making changes, it is for example possible that an amino acid residue belonging to a certain functional grouping of amino acid residues (e.g. hydrophobic, hydrophilic, polar etc) could be exchanged for another amino acid residue from the same functional group.

The term "% identity", as used throughout the specification, may for example be calculated as follows. The query sequence is aligned to the target sequence using the CLUSTAL W algorithm (Thompson et al, Nucleic Acids Research, 22: 4673-4680 (1994)). A comparison is made over the window corresponding to the shortest of the aligned sequences. The shortest of the aligned sequences may in some instances be the target sequence. In other instances, the query sequence may constitute the shortest of the aligned sequences. The amino acid residues at each position are compared, and the percentage of positions in the query sequence that have identical

correspondences in the target sequence is reported as % identity. In the alignment, deletions and additions of one or a few amino acid residues are suitably taken into account. When present, a deletion or addition of one amino acid residue in one of the aligned sequences is counted as one difference in the calculation of identity between the sequences. In one embodiment of the first aspect, there is provided a HER3 binding polypeptide, which is also capable of binding albumin. Albumin binding ability, when present in HER3 binding polypeptides of the present disclosure, is thought to arise as a consequence of retaining the original albumin-binding capacity of the G148-GA3 domain (or "ABDwt") and the stabilized version thereof ("ABD" and "ABD^*" in the Examples and Figures herein; "ABDmut" in WO00/23580) discussed in the Background section. It is advantageous for the HER3 binding polypeptide to bind to albumin because such albumin binding is expected to prolong the in vivo half-life of a

polypeptide, by for example avoiding rapid renal clearance. The relatively small HER3 binding polypeptide of the present disclosure, which is capable of binding to albumin in addition to HER3, is expected, due to its small size, not to be impaired in terms of vascular permeability and tumor penetration capability. This is in contrast to many significantly larger molecules.

In one embodiment of a polypeptide according to this aspect, X₃ in sequence i) is selected from R, S and T.

In one embodiment, X₃ in sequence s selected from S and T.

In one embodiment X3 in sequence i s selected from R and T.

In one embodiment X3 in sequence i s T.

In one embodiment X3 in sequence i s R.

In one embodiment X₃ n sequence s S.

In one embodiment X₆ n sequence s selected from A, K and R. In one embodiment X₆ n sequence s selected from K and R.

In one embodiment X₆ n sequence s R.

In one embodiment X₆ n sequence s K.

In one embodiment X₇ n sequence s selected from L and R.

In one embodiment X₇ n sequence In one embodiment Xg in sequence i) is L.

In one embodiment Xg in sequence i) is N.

In one embodiment Xio in sequence i) is selected from H and Y.

In one embodiment Xio in sequence i) is Y.

In one embodiment Xio in sequence i) is R.

In one embodiment Xi i in sequence i) is selected from F, I, L and V.

In one embodiment Xi i in sequence i) is selected from F, L and V.

In one embodiment Xi i in sequence i) is selected from I, L and V.

In one embodiment Xi i in sequence i) is selected from L and V.

In one embodiment Xi i in sequence i) is L.

In one embodiment Xi₄ in sequence i) is selected from A, D, G, K, L,

N, P, Q, R, S and T.

In one embodiment Xi₄ in sequence i) is selected from A, D, G, K, L,

N, P, Q, R and S.

In one embodiment Xi₄ in sequence i) is selected from D G, K, Q, R and S.

In one embodiment Xi₄ in sequence i) is selected from D G, K, R and

S.

In one embodiment Xi₄ in sequence i) is selected from G K, Q, R and

S.

In one embodiment Xi₄ in sequence i) is selected from D G, R and S.

In one embodiment Xi₄ in sequence i) is selected from G K, R, and S

In one embodiment Xi₄ in sequence i) is selected from G K and R.

In one embodiment Xi₄ in sequence i) is selected from G and R.

In one embodiment Xi₄ in sequence i) is selected from G and K.

In one embodiment Xi₄ in sequence i) is selected from S and R.

In one embodiment Xi₄ in sequence i) is G.

In one embodiment Xi₄ in sequence i) is R.

In one embodiment Xi₄ in sequence i) is K.

In one embodiment Xi₄ in sequence i) is S.

In one embodiment Xi₅ in sequence i) is selected from R T and V.

In one embodiment Xi₅ in sequence i) is selected from R and T.

In one embodiment Xi₅ in sequence i) is selected from R and V.

In one embodiment Xi₅ in sequence i) is selected from K and R.

In one embodiment Xi5 in sequence i) is R.

In one embodiment Xi₅ in sequence i) is T.

In one embodiment Xi₅ in sequence i) is V. In one embod ment Xl5 in sequence i ) is K.

In one embod ment X20 in sequence i ) is F.

In one embod ment X20 in sequence i ) is Y.

In one embod ment X23 in sequence i ) is D.

In one embod ment X23 in sequence i ) is R.

In one embod ment X35 in sequence i ) is selected from H, Q and R.

In one embod ment X35 in sequence i ) is selected from Q and R.

In one embod ment X35 in sequence i ) is selected from H, M and R.

In one embod ment X35 in sequence i ) is selected from H, M and Q.

In one embod ment X35 in sequence i ) is selected from H and M.

In one embod ment X35 n sequence i) is H.

In one embod ment X35 in sequence i ) is M.

In one embod ment X35 in sequence i ) is R.

In one embod ment X35 in sequence i ) is Q.

In one embod ment X38 in sequence i ) is selected from I, L, T and V.

In one embod ment X38 in sequence i ) is selected from I, V and L.

In one embod ment X38 in sequence i ) is selected from I and V.

In one embod ment X38 in sequence i ) is selected from I and L.

In one embod ment X38 in sequence i ) is selected from L and V.

In one embod ment X38 n sequence i) is I.

In one embod ment X38 in sequence i ) is L.

In one embod ment X38 in sequence i ) is selected from A, S, T and V.

In one embod ment X38 in sequence i ) is selected from S, T and V.

In one embod ment X38 in sequence i ) is V.

In one embod ment X38 in sequence i ) is selected from S and T.

In one embod ment X38 in sequence i ) is S.

In one embod ment X38 in sequence i ) is T.

In one embod ment X39 in sequence i ) is selected from F, I, L and R.

In one embod ment X39 in sequence i ) is selected from F, L and R.

In one embod ment X39 in sequence i ) is selected from L and R.

In one embod ment X39 in sequence i ) is L.

In one embod ment X39 in sequence i ) is R.

In one embod ment X40 in sequence i ) is A.

In one embod ment X40 in sequence i ) is E.

In one embod ment X43 in sequence i ) is selected from A, G, H, I, P, R

T and V. In one embodiment, X43 in sequence i) is selected from A, G, H, I, P, R and V.

In one embodiment, X43 in sequence is selected from A, G, R and V.

In one embodiment, X43 in sequence is selected from G, R and V.

In one embodiment, X43 in sequence is G.

In one embodiment, X43 in sequence is V.

In one embodiment, X43 in sequence is R.

In one embodiment, X43 in sequence is selected from A, I, H and P.

In one embodiment, X43 in sequence is selected from A, I and H.

In one embodiment, X43 in sequence is selected from A and I.

In one embodiment, X43 in sequence is A.

In one embodiment, X43 in sequence is I.

In one embodiment, X43 in sequence is H.

In one embodiment, sequence i) is

LATAKX₆LAX₉Y LLDRRGVSDX₂₀ YKX23LIDKAKT VEGVQALX₃₈RX₄o

wherein, independently of each other,

Χβ is selected from K and R;

Xg is selected from L and N;

X20 s selected from F and Y;

X23 s selected from D and R;

X38 s selected from I, L and V;

X40 s selected from A and E; and

X43 s selected from G, I, R and V. In a further embodiment of the HER3 binding polypeptide according to this aspect, sequence i) is selected from the group consisting of SEQ ID NO:1 -334 and 338-437, such as selected from the group consisting of SEQ ID NO:1 -334. In one embodiment, sequence i) is selected from the group consisting of SEQ ID NO:1 -167 and 338-387, such as selected from the group consisting of SEQ ID NO:1 -167. In another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:1 -160. In yet another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:1 -9, such as selected from SEQ ID NO:1 -4, such as selected from SEQ ID NO:1 -2. In one embodiment, said HER3 binding polypeptide is SEQ ID NO:1 . In another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:161 -167. In yet another embodiment, sequence i) is selected from SEQ ID NO:161 -164, such as selected from SEQ ID NO:161 - 162. In one embodiment, sequence i) is SEQ ID NO:161 . In another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:1 -9, 54, 83 and 86, such as selected from the group consisting of SEQ ID NO:4, 9, 54, 83 and 86.

In one embodiment of the polypeptide according to this aspect, sequence i) is selected from the group consisting of SEQ ID NO:168-334 and 388-437, such as selected from the group consisting of SEQ ID NO:168-334. In another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:168-327. In yet another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:168-176, such as selected from SEQ ID NO:168-171 , such as selected from SEQ ID NO:168-169. In one embodiment, sequence i) is SEQ IN NO:168. In another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:328-334. In yet another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:328-331 , such as selected from SEQ ID NO:328-329. In one embodiment, sequence i) is SEQ IN NO:328. In yet another embodiment, sequence i) is selected from the group consisting of SEQ ID NO:168-176, 221 , 250 and 253, such as selected from the group consisting of SEQ ID NO:171 , 176, 221 , 250 and 253.

The terms "HER3 binding" and "binding affinity for HER3" as used in this specification refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology. For example as described in the examples below, HER3 binding affinity may be tested in an experiment in which HER3, or a fragment thereof, is immobilized on a sensor chip of the instrument, and the sample containing the polypeptide to be tested is passed over the chip. Alternatively, the polypeptide to be tested is immobilized on a sensor chip of the instrument, and a sample containing HER3, or a fragment thereof, is passed over the chip. The skilled person may then interpret the results obtained by such experiments to establish at least a qualitative measure of the binding affinity of the polypeptide for HER3. If a quantitative measure is desired, for example to determine a K_D value for the interaction, surface plasmon resonance methods may also be used. Binding values may for example be defined in a Biacore (GE Healthcare) or ProteOn XPR36 (Bio-Rad) instrument. HER3 is suitably immobilized on a sensor chip of the instrument, and samples of the polypeptide whose affinity is to be determined are prepared by serial dilution and injected in random order. K_D values may then be calculated from the results using for example the 1 :1 Langmuir binding model of the BIAevaluation 4.1 software, or other suitable software, provided by the instrument manufacturer.

The terms "albumin binding" and "binding affinity for albumin" as used in this specifications refer to a property of a polypeptide which may be tested for example by the use of surface plasmon resonance technology, such as in a Biacore instrument or ProteOn XPR36 instrument, in an analogous way to the example described above for HER3. In one embodiment, the HER3 binding polypeptide is capable of binding to HER3 such that the K_D value of the interaction is at most

1 x 10^"8 M, such as at most 1 x 10^"9 M, such as at most 1 x 10^"10 M, such as at most 1 x 10^{"1 1} M.

In one embodiment of those polypeptides disclosed herein that have a dual affinity for both HER3 and albumin, the HER3 binding polypeptide is capable of binding to albumin such that the K_D value of the interaction is at least 1 x 10^"8 M, such as at least 1 x 10^"7 M, such as at least 1 x 10^"6 M, such as at least 1 x 10^"5 M. In one embodiment, the K_D value of the interaction with albumin is within the range from 1 x 10^{"1 1} M to 1 x 10^"6 M, such as within the range from 1 x 10^"9 M to 1 x 10^"6 M.

In one embodiment of those polypeptides disclosed herein that have a dual affinity for both HER3 and albumin, the affinity of the HER3 binding polypeptide to HER3 is higher than its affinity for albumin, such that the HER3 binding polypeptide is preferentially bound to HER3 in the presence of both HER3 and albumin, but binds to albumin in the absence of HER3.

In one embodiment, said albumin is human serum albumin.

The skilled person will understand that various modifications and/or additions can be made to a HER3 binding polypeptide according to any aspect disclosed herein in order to tailor the polypeptide to a specific application without departing from the scope of the present disclosure. For example, any HER3 binding polypeptide disclosed herein may comprise further C terminal and/or N terminal amino acids. Such a

polypeptide should be understood as a polypeptide having additional amino acid residues at the very first and/or the very last position in the polypeptide chain, i.e. at the N- and/or C-terminus. Thus, a HER3 binding polypeptide may comprise any suitable number of additional amino acid residues, for example at least one additional amino acid residue. Each additional amino acid residue may individually or collectively be added in order to, for example, improve production, purification, stabilization in vivo or in vitro, coupling, or detection of the polypeptide. Such additional amino acid residues may comprise one or more amino acid residues added for the purpose of chemical coupling. One example of this is the addition of a cysteine residue. Such additional amino acid residues may also provide a "tag" for purification or detection of the polypeptide, such as a His6 tag or a "myc" (c-myc) tag or a "FLAG" tag for interaction with antibodies specific to the tag or immobilized metal affinity chromatography (IMAC) in the case of the His6-tag.

The further amino acids as discussed above may be coupled to the HER3 binding polypeptide by means of chemical conjugation (using known organic chemistry methods) or by any other means, such as expression of the HER3 binding polypeptide as a fusion protein or joined in any other fashion, either directly or via a linker, for example an amino acid linker.

The further amino acids as discussed above may for example comprise one or more polypeptide domain(s). A further polypeptide domain may provide the HER3 binding polypeptide with another function, such as for example another binding function, or an enzymatic function, or a toxic function (e.g. an immunotoxin), or a fluorescent signaling function, or combinations thereof.

A further polypeptide domain may moreover provide the HER3 binding polypeptide with the same binding function. Thus, in a further embodiment, there is provided a HER3 binding polypeptide as a multimer, such as dimer. Said multimer is understood to comprise at least two HER3 binding

polypeptides as disclosed herein as monomer units, the amino acid

sequences of which may be the same or different. Multimeric forms of the polypeptides may comprise a suitable number of domains, each having a HER3 binding motif, and each forming a monomer within the multimer. These domains may have the same amino acid sequence, but alternatively, they may have different amino acid sequences. In other words, the HER3 binding polypeptide of the invention may form homo- or heteromultimers, such as homo- or heterodimers.

Alternatively, one or more further polypeptide domain(s) may provide the HER3 binding polypeptide with another binding function. Thus, in a further embodiment, there is provided a HER3 binding polypeptide comprising at least one HER3 binding polypeptide monomer unit and at least one monomer unit with a binding affinity for another target. Such other target may

advantageously be selected from other epidermal growth factor receptors, such as in particular HER2. Such heteromultimeric forms of the polypeptide may comprise a suitable number of domains, having at least one HER3 binding motif, and a suitable number of domains with binding motifs conferring affinity to the other target. Additionally, "heterogenic" fusion polypeptides or proteins, or conjugates, in which a HER3 binding polypeptide according to the disclosure, or multimer thereof, constitutes a first domain, or first moiety, and the second and further moieties have other functions than binding HER3, are also contemplated and fall within the ambit of the present invention. The second and further moiety/moieties of the fusion polypeptide or conjugate in such a protein suitably have a desired biological activity.

Thus, in a second aspect of the present invention, there is provided a fusion protein or a conjugate, comprising a first moiety consisting of a HER3 binding polypeptide according to the first aspect, and a second moiety consisting of a polypeptide having a desired biological activity. In another embodiment, said fusion protein or conjugate may additionally comprise further moieties, comprising desired biological activities that can be either the same or different from the biological activity of the second moiety.

Non-limiting examples of such a desired biological activity comprise a therapeutic activity, a binding activity, and an enzymatic activity. In one embodiment, the second moiety having a desired biological activity is a therapeutically active polypeptide.

Non-limiting examples of therapeutically active polypeptides are biomolecules, such as molecules selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines. In one embodiment of this aspect of the present disclosure, there is provided a HER3 binding polypeptide, fusion protein or conjugate which further comprises a cytotoxic agent. Non-limiting examples of cytotoxic agents are agents selected from the group consisting of auristatin, anthracycline, calicheamycin, combretastatin, doxorubicin, duocarmycin, the CC-1065 anti- tumorantibiotic, ecteinsascidin, geldanamycin, maytansinoid, methotrexate, mycotoxin, ricin and its analogues, taxol and derivates thereof and

combinations thereof.

As the skilled person understands, the HER3 binding polypeptide according to the first aspect may be useful in a fusion protein or as a conjugate partner to any other moiety. Therefore, the above lists of therapeutically active polypeptides and cytotoxic agents should not be construed as limiting in any way.

Other possibilities for the creation of fusion polypeptides or conjugates are also contemplated. Thus, an HER3 binding polypeptide according to the first aspect of the invention may be covalently coupled to a second or further moiety or moieties, which in addition to or instead of target binding exhibit other functions. One example is a fusion between one or more HER3 binding polypeptide(s) and an enzymatically active polypeptide serving as a reporter or effector moiety.

With regard to the description above of fusion proteins or conjugates incorporating a HER3 binding polypeptide according to the invention, it is to be noted that the designation of first, second and further moieties is made for clarity reasons to distinguish between HER3 binding polypeptide or polypeptides according to the invention on the one hand, and moieties exhibiting other functions on the other hand. These designations are not intended to refer to the actual order of the different domains in the polypeptide chain of the fusion protein or conjugate. Thus, for example, said first moiety may without restriction appear at the N-terminal end, in the middle, or at the C-terminal end of the fusion protein or conjugate.

The above aspects furthermore encompass polypeptides in which the HER3 binding polypeptide according to the first aspect, or the HER3 binding polypeptide as comprised in a fusion protein or conjugate according to the second aspect, further comprises a label, such as a label selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles. Such labels may for example be used for detection of the polypeptide. For example, in some embodiments such labeled polypeptide may for example be used for indirect labeling HER3 expressing tumors cells as well as metastatic cells.

In other embodiments, the labeled HER3 binding polypeptide is present as a moiety in a fusion protein or conjugate also comprising a second moiety having a desired biological activity. The label may in some instances be coupled only to the HER3 binding polypeptide, and in some instances both to the HER3 binding polypeptide and to the second moiety of the conjugate or fusion protein. Furthermore, it is also possible that the label may be coupled to a second moiety only and not the HER3 binding moiety. Hence, in yet another embodiment, there is provided an HER3 binding polypeptide comprising a second moiety, wherein said label is coupled to the second moiety only.

When reference is made to a labeled polypeptide, this should be understood as a reference to all aspects of polypeptides as described herein, including fusion proteins and conjugates comprising a HER3 binding polypeptide and a second and optionally further moieties. Thus, a labeled polypeptide may contain only the HER3 binding polypeptide and e.g. a therapeutic radionuclide, which may be chelated or covalently coupled to the HER3 binding polypeptide, or contain the HER3 binding polypeptide, a therapeutic radionuclide and a second moiety such as a small molecule having a desired biological activity, for example a therapeutic efficacy.

In embodiments where the HER3 binding polypeptide, fusion protein or conjugate is radiolabeled, such a radiolabeled polypeptide may comprise a radionuclide. A majority of radionuclides have a metallic nature and metals are typically incapable of forming stable covalent bonds with elements presented in proteins and peptides. For this reason, labeling of proteins and peptides with radioactive metals is performed with the use of chelators, i.e. multidentate ligands, which form non-covalent compounds, called chelates, with the metal ions. In an embodiment of the HER3 binding polypeptide, fusion protein or conjugate, the incorporation of a radionuclide is enabled through the provision of a chelating environment, through which the

radionuclide may be coordinated, chelated or complexed to the polypeptide.

One example of a chelator is the polyaminopolycarboxylate type of chelator. Two classes of such polyaminopolycarboxylate chelators can be distinguished: macrocyclic and acyclic chelators. In one embodiment, the HER3 binding polypeptide, fusion protein or conjugate comprises a chelating environment provided by a

polyaminopolycarboxylate chelator conjugated to the HER3 binding polypeptide via a thiol group of a cysteine residue or an epsilon amine group of a lysine residue.

The most commonly used macrocyclic chelators for radioisotopes of indium, gallium, yttrium, bismuth, radioactinides and radiolanthanides are different derivatives of DOTA (1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10- tetraacetic acid). In one embodiment, a chelating environment of the HER3 binding polypeptide, fusion protein or conjugate is provided by DOTA or a derivative thereof. More specifically, in one embodiment, the chelating polypeptides encompassed by the present disclosure are obtained by reacting the DOTA derivative 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tris- acetic acid-10-maleimidoethylacetamide (maleimidomonoamide-DOTA) with said polypeptide.

Additionally, 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA) and derivatives thereof may be used as chelators. Hence, in one embodiment, there is provided a HER3 binding polypeptide, fusion protein or conjugate, wherein the the polyaminopolycarboxylate chelator is 1 ,4,7- triazacyclononane-1 ,4,7-triacetic acid or a derivative thereof.

The most commonly used acyclic polyaminopolycarboxylate chelators are different derivatives of DTPA (diethylenetriamine-pentaacetic acid).

Hence, polypeptides having a chelating environment provided by

diethylenetriaminepentaacetic acid or derivatives thereof are also

encompassed by the present disclosure.

In a third aspect of the present invention, there is provided a polynucleotide encoding a HER3 binding polypeptide or a fusion protein as described herein.

Also encompassed by this disclosure is a method of producing a polypeptide or fusion protein as described above, comprising expressing a polynucleotide; an expression vector comprising the polynucleotide; and a host cell comprising the expression vector.

Also encompassed is a method of producing a polypeptide, comprising culturing said host cell under conditions permissive of expression of said polypeptide from its expression vector, and isolating the polypeptide. The HER3 binding polypeptide of the present disclosure may

alternatively be produced by non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side-chains, the non-biological peptide synthesis comprising

- step-wise coupling of the amino acids and/or the amino acid derivatives to form a polypeptide according to the first aspect having protected reactive side-chains,

- removal of the protecting groups from the reactive side-chains of the polypeptide, and

- folding of the polypeptide in aqueous solution.

It should be understood that the HER3 binding polypeptide according to the present disclosure may be useful as a therapeutic or diagnostic agent in its own right or as a means for targeting other therapeutic or diagnostic agents, with e.g. direct or indirect effects on HER3. A direct therapeutic effect may for example be accomplished by inhibiting HER3 signaling.

In another aspect, there is provided a composition comprising a HER3 binding polypeptide, fusion protein or conjugate as described herein and at least one pharmaceutically acceptable excipient or carrier. In one

embodiment thereof, the composition further comprises at least one additional active agent, such as at least two additional active agents, such as at least three additional active agents. Non-limiting examples of additional active agents that may prove useful in such a combination are immunostimulatory agents, radionuclides, toxic agents, enzymes, factors recruiting effector cells (e.g. T or NK cells) and photosensitizers.

In one embodiment of the present invention, there is provided a HER3 binding polypeptide, fusion protein, conjugate or composition as described herein for use as a medicament. Also, in another embodiment there is provided a HER3 binding polypeptide, fusion protein .conjugate or

composition as described herein for use in diagnosis. In one embodiment, there is provided a HER3 binding polypeptide, fusion protein, conjugate or composition for use in treatment or diagnosis of HER3 related condition, such as cancer.

The term "cancer" or hyperproliferative disease as used herein refers to tumor diseases and/or cancer, such as metastatic or invasive cancers, for example lung cancer, non small cell lung (NSCL) cancer, bronchioloalviolar cell lung cancer, bone cancer, pancreatic cancer, skin cancer, cancer of the head or neck, cutaneous or intraocular melanoma, rectal cancer, cancer of the anal region, stomach cancer, gastric cancer, colon cancer, colorectal cancer, cancer of the small intestines, esophageal cancer, liver cancer, pancreas cancer, breast cancer, ovarian cancer, uterine cancer, carcinoma of the fallopian tubes, carcinoma of the endometrium, carcinoma of the cervix, carcinoma of the vagina, carcinoma of the vulva, Hodgkin's Disease, cancer of the endocrine system, cancer of the thyroid gland, cancer of the

parathyroid gland, cancer of the adrenal gland, sarcoma of soft tissue, cancer of the urethra, cancer of the penis, prostate cancer, bladder cancer, cancer of the kidney or ureter, renal cell carcinoma, carcinoma of the renal pelvis, mesothelioma, hepatocellular cancer, biliary cancer, neoplasms of the central nervous system (CNS), spinal axis tumors, brain stem glioma, glioblastoma multiforme, astrocytomas, schwanomas, ependymonas, medulloblastomas, meningiomas, squamous cell carcinomas, pituitary adenoma, lymphoma, lymphocytic leukemia, or cancer of unknown origin, or other hyperplastic or neoplastic HER3 related condition, including refractory versions of any of the above cancers or a combination of one or more of the above cancers or hyperproliferative diseases.

In one embodiment, said HER3 related condition or cancer, is further characterized by HER3 expression, over-expression and/or activation, e.g. hyperphosphorylation. Non limiting examples of HER3 related conditions are cancers selected from the group consisting of lung cancer, breast cancer, gastric cancer, stomach cancer, colon cancer, colorectal cancer, cancer of the small instestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, melanomas, cancers of the head and neck, pediatric gliomas, pediatric glioblastomas and astrocytomas. In a related aspect, there is provided a method of treatment of a HER3 related condition, comprising administering to a subject in need thereof an effective amount of a HER3 binding polypeptide, fusion protein, conjugate or composition as described herein. Consequently, in the method of treatment, the subject is treated with a HER3 binding polypeptide or a HER3 binding combination according to the invention. In a more specific embodiment of said method, the HER3 binding polypeptide, fusion protein, conjugate or composition inhibits HER3 mediated signaling by binding to HER3 expressed on a cell surface. In one embodiment of said method, the HER3 related condition is cancer. In one embodiment of said method, the cancer is selected from the group consisting of lung cancer, breast cancer, gastric cancer, stomach cancer, colon cancer, colorectal cancer, cancer of the small instestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, melanomas, cancers of the head and neck, pediatric gliomas, pediatric glioblastomas and astrocytomas. While the invention has been described with reference to various exemplary aspects and embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be

substituted for elements thereof without departing from the scope of the invention. In addition, many modifications may be made to adapt a particular situation or molecule to the teachings of the invention without departing from the essential scope thereof. Therefore, it is intended that the invention not be limited to any particular embodiment contemplated, but that the invention will include all embodiments falling within the scope of the appended claims. Brief description of the figures

Throughout the Examples and Figures, the synonym term "ErbB3" is used interchangeably with the term "HER3".

Figure 1 is a listing of the amino acid sequences of examples of HER3 binding polypeptides according to the disclosure (SEQ ID NO:1 -334 and 338- 437) as well the HER3 binding Z variant Z05417 (SEQ ID NO:336), alkali stabilized G148-GA3 (SEQ ID NO:335; in the Examples and Figures denoted

"ABD" or "ABD^*"; see Background section above and PCT publication

WO00/23580, in which the same polypeptide is denoted "ABDmut") and wild type G148-GA3 (SEQ ID NO:337).

Figure 2A shows a molecular model of G148-GA3 (SEQ ID NO:337)

(PDB file name 1 GJS).

Figure 2B is a schematic overview of the phage display selection of

HER3 binding ABD variants as described in Example 1 . Four cycles (1 -4) of phage display was performed with increasing selection pressure in each round. This was achieved by decreasing the HER3 concentration in parallel tracks and increasing the number of washes prior to elution. Figure 2C is a multi-alignment tree of the amino acid sequences of clones recovered after four rounds of selection showing sequence similarity represented by branch lengths (solid lines), which are proportional to the number of differing amino acids measured as average number of mutations per residue. The number of times a clone was observed during sequencing and track origin is displayed to the right.

Figure 2D shows the amino acid sequences of seven clones (SEQ ID NO:161 -167) recovered after four rounds of selection as described in

Example 1 and ABD (SEQ ID NO:335). Predicted helices 1 , 2 and 3 are indicated in boxes.

Figure 3A shows representative SPR sensorgrams for ABD3-3 (SEQ ID NO:161 ) and ABD3-27 (SEQ ID NO:162) binding to human serum albumin (HSA) and hHER3.

Figure 3B shows overlaps of circular dichroism spectra for ABD3-3 (SEQ ID NO:161 ) and ABD3-27 (SEQ ID NO:162) before and after thermal denaturation (TD).

Figure 4 shows the result of binding of the indicated ABD variants ABD3-3, ABD3-18, ABD3-20 and ABD3-27 (SEQ ID NO;161 -164) to hHER3 in the presence of albumin, as described in Example 4. HER3 binding ABD variants were incubated with varying concentrations of albumin for at least 1 h at room temperature and subsequently injected over a sensor chip with immobilized HER3. Data in Figure 4A and Figure 4B were collected from two separate experiments using different sensor chips. 500 nM ABD was used in all incubations which were injected over one surface with hHER3 and two surfaces with mHER3 in the experiment shown in Figure 4A. 200 nM ABD was used in all incubations which were injected over two surfaces with hHER3 in the experiment shown in Figure 4B. All observed responses were normalized against reference injections performed in each respective experiment. Data bars represent mean values from 3 (Figure 4A) and 4 (Figure 4B) interactions in total (error bars: ±SD).

Figure 5 shows the result of binding experiments in which 5 nM hHER3 was incubated with the indicated concentrations of test molecules, and injected over a sensor chip with immobilized NRG-βΙ ECD as described in Example 5. Obtained max responses were normalized against a

corresponding injection with untreated hHER3. Bars represent mean values with indicated standard deviations from two injections over three surfaces (n=6). Control injection of NRG-βΙ was performed only once (n=3). Figure 6A shows the result of binding of the indicated ABD variants to HER3-expressing cells in vitro as described in Example 6. Bars represent mean values from multiple measurements performed on different days (error bars: ±SD). The background was measured from cells treated in the same manner with the ABD omitted. All MFI-values were normalized by subtracting the mean autofluorescent intensity measured from cells treated with PBSTB only.

Figure 6B is a bar graph showing MRI values for ABD-Z or (ABD)₂-Z when AU565 cells were additionally pre-incubated with or without 100 nM NRG-βΙ (heregulin).

Figure 7 shows a table representing the design of the affinity maturation library as described in Example 7. The resulting theoretical relative frequency (0 = 0 %, 0.5 = 50 %) of each amino acid is given for each randomized codon position. The amino acids are ordered after approximate decreasing polarity at physiological pH, from charged R to hydrophobic Y.

Figure 8A is a pie chart illustrating that 575 (88 %) of the 651 sequenced single colonies contained an ABD variant sequence.

Figure 8B is a pie chart illustrating the distribution of said 575 ABD sequences into full-length, sequences containing deletion(s), incomplete sequences, sequences containing multiple errors (mutation/insertion/ deletion), sequences containing mutation(s) and sequences containing insert(s).

Figure 8C is a table representing in silico translation of the observed 217 full-length sequences at each randomized codon position expressed as relative frequency where 0 is found in no sequences and 0.6 is found in 60 % of sequences. The amino acids are ordered after approximate decreasing polarity at physiological pH, from charged R to hydrophobic Y.

Figure 8D is a table illustrating the difference in relative frequency between the designed library in theory and the observed sequences at each randomized codon position, calculated by subtracting the observed from the theoretical relative frequency as shown in Figure 7. A negative value indicates a lower observed prevalence while a positive indicates an increased observed prevalence compared to the design. All expected amino acids were observed on the randomized positions and no difference in relative frequency larger than 0.15 was observed.

Figure 9 shows a schematic illustration of flow cytometric cell sorting (FACS) of library cells as used herein as described in Example 8. Figure 10 shows scatter plots of cell populations expressing the unsorted library and the populations after round 1 , 2 and 3 of FACS-based sorting of HER3 binding polypeptides (using PE-labeled HER3)

demonstrating variability in HER3 binding and IgG binding properties.

Enrichment of HER3 binding polypeptides is shown over the sorting rounds. Three subsequent rounds of FACS-based sorting were performed at 50 nM, at 10 nM and at 10 nM, 1 .0 nM or 0.1 nM HER3 and gating was performed as indicated. Scatter plots of cell populations expressing ABD (SEQ ID NO:335), HER3-3 (ABDHERS-S) (SEQ ID NO:161 ) and Z05417 (SEQ ID NO:336) are shown for comparison or as controls. HER3 binding is shown on the y-axis and expression levels, reported using fluorescently labeled IgG binding to the Z2-domain, are shown on the x-axis.

Figure 1 1 shows a sequence logotype representing the unsorted affinity maturation library, constructed as described in Example 7. The eleven randomized positions are shown from left (N-terminus) to right (C-terminus) and their locations in the 46 amino acid sequence are indicated by numbers.

Figure 12 shows sequence logotypes representing affinity matured HER3 binding ABD variants after three rounds of flow-cytometric sorting as described in Example 9, wherein the HER3 concentration in the third round was 0.1 nM (Figure 12A), 1 nM (Figure 12B) or 10 nM (Figure 12C). The eleven randomized positions are shown from left (N-terminus) to right (C- terminus) and their locations in the 46 amino acid sequence are indicated by numbers.

Figure 13 shows a sequence logotype representing all affinity matured HER3 binding ABD variants after three rounds of sorting as described in Example 9. The eleven randomized positions are shown from left (N- terminus) to right (C-terminus) and their locations in the 46 amino acid sequence are indicated by numbers.

Figure 14 shows a sequence logotype representing the nine most frequent affinity matured HER3 binding ABD variants (SEQ ID NO:1 -9). All 46 positions are shown from left (N-terminus) to right (C-terminus). The nine HER3 binding ABD variants are listed, and the differences between their amino acid sequences and the consensus sequence are indicated.

Figure 15 shows an overview of all the flow cytometric selections performed. The sorting gates used in each selection round are indicated and the percentage of sorted cells of the total number of interrogated cells is indicated within the gates. The post-selection analyses of outputs were performed with 1 nM biotinylated HER3 incubated to equilibrium. All y-axis parameters are log-scale fluorescence intensities measured from SA-PE, and all x-axis parameters are log-scale fluorescence intensities measured from lgG-647.

Figure 16 shows the results of flow-cytometric screening of 165 variants as described in Example 10. Median fluorescence intensities of detected HER3 were normalized against the expression level measured as median fluorescence intensities of fluorescent IgG. A) Result of screening all 165 variants. B) Ranking of 28 clones with the highest binding signal and sequence frequency. C) Additional screening of clones at 50 nM HER3 to confirm positive HER3-binding. Controls are indicated as gray bars, which include ABD_HER3-3 and non-randomized ABD.

The invention will now be illustrated further through the non-limiting description of experiments conducted in accordance therewith. Unless otherwise specified, conventional chemistry and molecular biology methods were used throughout.

Examples

As used throughout the example section of this specification, HER3 binding polypeptides according to the invention are referred to according to the ABD3-N nomenclature, wherein N is an integer. Also, herein the HER3 binding polypeptides according to the invention are referred to as ABD variants.

Summary

The Examples which follow disclose the development of novel, bi- specific single domain binders targeted towards HER3 and albumin, referred to herein as HER3 binding polypeptides or HER3 binding ABD variants, based on phage display technology and flow-cytometry based sorting. The HER3 binding polypeptides described herein were sequenced, and their amino acid sequences are listed in Figure 1 with the sequence identifiers SEQ ID NO:1 -167 and 338-387. Also, the deduced binding motifs of these selected binding variants were incorporated by deduction into a previously described optimized albumin binding domain sequence, generating definitions of ABD variants listed in Figure 1 with sequence identifiers SEQ ID NO:168- 334 and 388-437. Briefly, in the following disclosure, the inventors develop bi-specific, single domain binders targeted towards HER3 and albumin, based on phage display selection from a combinatorial library of a starting albumin binding domain from streptococcal protein G. The inventors show that the intrisic albumin binding properties can be combined, within a single 5 kDa domain, with the ability to bind with high affinity to HER3 on human cells in culture. The inventors disclose a set of seven first generation HER3 binding polypeptides (SEQ ID NO:161 -167), and demonstrate that these HER3 binding polypeptides compete for binding with heregulin, a natural HER3 ligand which stimulates cell proliferation in an affinity dependent manner.

Furthermore, the inventors continue by designing an affinity maturation library based on the sequences of the HER3 binding polypeptides identified by phage display and express this library in the bacterial strain Staphylococcus carnosus to perform flow-cytometric sorting to isolate a set of second generation HER3 binding polypeptides (SEQ ID NO:1 -160). After this experiment, which is performed twice yielding very similar results, an additional sorting based on off-rate characteristics is performed, which yields an additional 50 unique, HER3 binding polypeptides (SEQ ID NO:338-387).

Example 1

Phage display selection, expression and purification of HER3 binding ABD variants Summary

This example describes four cycles of phage display selection, carried out with an increasing selection pressure in each round, performed in order to identify ABD polypeptide variants with a specificity for HER3. The increase in selection pressure was achieved by decreasing the HER3 concentration in parallel tracks and increasing the number of washes prior to elution. 32 unique HER3 binding polypeptides were identified, which exhibit binding affinity for both HER3 and albumin. Seven of these were characterized in greater detail. These seven HER3 binding polypeptides were sequenced and their amino acid sequences are listed with sequence identifiers SEQ ID NO:161 -167 in Figure 1 . Materials and methods

HER3 binding molecules were selected by phage display from a combinatorial library based on an albumin-binding domain where eleven surface exposed residues not directly involved in albumin binding were randomized. Escherichia coli (E. coli) RR1 AM15 strain (Ruther et al, Nucleic acids research 10 (19):5765-5772 (1982)) carrying the phagemid library (Aim et al, Biotechnology journal 5 (6):605-617 (2010)) was cultured by inoculating 500 ml tryptic soy broth (TSB) supplemented with 2 % (w/v) glucose and 100 g/ml ampicillin with 100 μΙ bacterial stock with a cell density of 2.2 x 10¹⁰ cfu/ml, corresponding to an approximate 100-fold excess of bacteria compared to the experimentally determined library size (Aim T el al, supra). In cycles 3 and 4, the culture volume was reduced to 100 ml. The number of cells used for inoculation was always at least a 10⁴-fold excess compared to the number of phages eluted from the previous cycle to ensure retained coverage. A 15-fold excess of M13K07 helper-phage (New England Biolabs (NEB), Ipswich, MA, USA) was allowed to infect a 8 ml culture (4 ml in cycle 3 and 4) aliquot during a still 2 h incubation at 37 °C. Cells were harvested by centrifugation and used to inoculate 500 ml TSB medium (100 ml for cycles 3 and 4) supplemented with 5 % (w/v) yeast extract (TSB+Y), 100 g/ml ampicillin, 100 g/ml kanamycin and 1 mM isopropyl β-D-l - thiogalactopyranoside (IPTG, Apollo Scientific, Derbyshire, United Kingdom) followed by overnight incubation at 30 °C to produce library-expressing phage.

A phage stock was prepared by two successive precipitation steps using polyethylene glycol (PEG)/NaCI followed by re-suspension in 1 ml phosphate buffered saline (PBS) pH 7.4 supplemented with 3 % (w/v) BSA and 0.05 % (v/v) Tween 20 (3 % PBSTB). As a negative pre-selection step, the phages were incubated for 30 min with 100 nM of purified human polyclonal IgG-Fc (Bethyl Laboratories, Montgomery, TX, USA) and 0.6 mg of Dynabeads® Protein A magnetic beads (Invitrogen, Carlsbad, CA, USA) that had been washed twice with 500 μΙ PBS supplemented with 0.1 % Tween 20 (PBST) and blocked for 20 min with 500 μΙ PBS supplemented with 0.1 % (v/v) Tween 20 and 5 % (w/v) BSA (5 % PBSTB). For the first selection cycle, IgG-Fc was not included during pre-selection. The entire phage-stock (10¹¹ phages) was used in cycle 1 and a 10⁴-fold excess compared to the number of previously eluted phages was used in subsequent cycles. The unbound phages were recovered in the supernatant, transferred to a new tube and incubated for 2 h with recombinant human HER3-Fc chimera (cat no. 348-RB R&D Systems, Minneapolis, MN, USA) before being transferred to 1 .5 mg of washed Dynabeads® Protein A (0.6 mg in cycle 3 and 4). Beads from both pre-selection and selection were washed with PBST as indicated in Figure 2B, and the bound phages were eluted from the beads by incubation with 500 μΙ 50 mM glycine-HCI pH 2.7 for 10 min. The eluate was immediately neutralized upon transfer to a new tube containing an equal volume of PBS with 10 % (v/v) 1 M Tris-HCI pH 8.0. The eluate was used to infect a fresh culture of RRIAM15 grown to OD600 ~ 0.5 and incubated still at 37 °C for 30 min. Infected cells were harvested by centrifugation, re-suspended in TSB+Y and spread on tryptone yeast extract (TYE) agar plates with

100 g/ml ampicillin and 2 % (w/v) glucose and incubated at 37 °C over night. Colonies were harvested and used to produce phages for the next round of selection. During selection, samples were collected from the last washes and eluates of both negative and positive selection, and used to infect RR1AM15 to determine phage titers. A total of four cycles of selection were performed with decreasing concentration of target and increasing number of washes according to Figure 2B. All micro-centrifuge tubes used were blocked with 500 μΙ of 5 % PBSTB end over end for at least 1 h. All steps were performed at room temperature and tubes were incubated end over end during incubations.

ABD variant inserts in colonies originating from the fourth cycle of selection were amplified by polymerase chain-reaction (PCR), sequenced by Sanger sequencing and analyzed on an ABI Prism 3700 DNA analyzer (Applied Biosystems, Foster City, CA, USA). Phagemids from colonies of interest were purified from small-scale cultivations using a plasmid purification kit (Qiagen, Solna, Sweden) and used as templates for PCR amplification of the ABD variant genes. PCR fragments were restricted with EcoRI and Xhol (NEB) and ligated into an expression vector with a T7 promoter and an N- terminal His6-tag that had been treated with the same enzymes,

dephosphorylated with alkaline phosphatase (NEB) and purified from a 1 % agarose gel with a DNA gel extraction kit (Qiagen). Ligated vectors were transformed to RRIAM15 and grown on TYE agar supplemented with

50 g/ml kanamycin. Single colonies were picked for PCR amplification and sequence verification. Plasmids carrying the correct constructs were purified and transformed to Rosetta (DE3) E. coli (Novagen, Wl, USA) for protein expression. Single colonies of Rosetta with ABD variant sequences cloned into the expression vector were used to inoculate overnight cultures in TSB

supplemented with 50 g/ml kanamycin and 20 g/ml chloramphenicol. 1 ml of the culture was used to inoculate 100 ml of TSB+Y supplemented with the same antibiotics and cultured at 37°C to OD₆oo ^s 0.5. The seven variants (SEQ ID NO:161 -167) chosen for sub-cloning and expression together with the non-randomized scaffold ABD (SEQ ID NO:335) as control are indicated in Figure 2D. Protein expression was induced by addition of IPTG to a final concentration of 1 mM and cultures were incubated overnight at 25 °C. Cells were harvested by centrifugation and re-suspended in Tris-buffered saline (TST; 25 mM Tris-HCI, 200 mM NaCI, 1 mM EDTA, 0.05 % (v/v) Tween 20, pH 8). Cells were lysed by sonication using a Vibra-Cell (Sonics & materials inc., Newtown, CT, USA), and cell debris was removed by an additional centrifugation. The supernatants were filtered prior to loading onto a resin with human serum albumin (HSA)-sepharose, with column volumes (CV) of 7.5 ml, pre-equilibrated with TST. Following sample loading, the columns were washed with 10 CV of TST followed by 7 CV of 5 mM NH₄Ac pH 5.5 and the proteins were eluted in fractions of 1 ml in 0.5 M HAc pH 2.8. Protein- containing fractions (as determined by absorbance at 280 nm) were evaporated over night using a Savant AES2010 SpeedVac system (Thermo Scientific, Rockford, IL, USA). Proteins were dissolved in PBST and analyzed by sodium dodecyl sulphate polyalycramide gel electrophoresis (SDS-PAGE) and mass spectrometry on a 6520 Accurate Q-TOF LC/MS instrument (Agilent, Santa Clara, CA, USA). Exact protein concentrations were determined from amino acid sequences and amino acid analysis

(Aminosyraanalyscentralen, Uppsala University, Sweden).

Results

In order to select molecules having specificity for HER3, four rounds of selection were performed using a recombinant form of the extracellular human receptor fused to human lgG1 -Fc as a target (Figure 2B). Phages were pre-incubated with polyclonal human lgG1 -Fc in a negative selection step prior to positive selection against HER3. After four rounds of selection, 32 unique ABD variants (ABD3-1 to ABD3-32) with an overall high sequence similarity were observed among the 136 clones that were sequenced. Two clones were observed more frequently; ABD3-1 (SEQ ID NO:165) was found 48 times and ABD3-2 (SEQ ID NO:166) 33 times in multiple selection tracks, whereas the remaining sequences appeared 1 -7 times each in the data set. Multi-alignment of all sequences revealed many similarities and patterns of conservation for a number of the residues that were randomized in the library. Seven individual clones (SEQ ID NO:161 -167) (Figure 2C), representing different clusters of the selection output, were sub-cloned into an expression vector and expressed in E. coli for further characterization. All expressed proteins were purified by HSA-affinity chromatography, which confirmed the retained affinity to albumin for the selected clones. High purity was obtained, as confirmed by SDS-PAGE, and expected molecular weights from the amino acid sequences were confirmed for all purified proteins by mass

spectrometry.

Example 2

Circular dichroism spectroscopy

Materials and methods

The secondary structure content and thermal stability of selected ABD variants was assessed by circular dichroism (CD) using a Jasco J-810

Spectropolarimeter (Jasco, Essex, United Kingdom). Proteins were buffer exchanged to PBS using NAP-5 gel filtration columns (GE Healthcare, Uppsala, Sweden) according to the manufacturer's recommendations and diluted to 0.4 mg/ml. Measurements were carried out in three steps; first, secondary structure content was assessed by measuring the degree of ellipticity from 250 nm to 195 nm. Second, the ellipticity at 221 nm was measured during heating of the sample from 25 °C to 90 °C to find the melting temperature (T_m). Lastly, spectra were again recorded from 250 nm to 195 nm at 25 °C to verify proper refolding of the proteins. Results

All analyzed variants had similar spectral curves with two distinct local extreme points at around 208 and 221 nm, similar to what has been shown earlier for ABD and G148-GA3 (Linhult et al, Protein Society 1 1 (2)206-213 (2002), Kraulis et al, FEBS letters 378 (2):190-194 (1996); Gulich et al, Journal of biotechnology 80 (2):169-178 (2000)). For almost all protein variants, the spectra before and after heat treatment overlapped well, which indicates that the proteins refold upon cooling to ambient temperature. As evident from Figure 3B, the only variant that did not completely refold after heating was ABD3-27 (SEQ ID NO:162). Interestingly, this binder exhibited a deviating denaturation profile with an intermediate plateau at 70 °C followed by an increasing CD signal that, at the highest temperature (90 °C), had not reached complete denaturation. Complete unfolding was not observed for any of the protein domains, even after heating to 90 °C. Hence, the T_m values could not be exactly defined from the thermal denaturation spectra at 221 nm. However, the values could be estimated to exceed 60 °C for some of the variants and to more than 80 °C for others (see listing in Table 1 in Example 3 below).

Example 3

Analysis of binding kinetics

Materials and methods

Binding kinetics for the seven selected ABD variants to albumin and HER3 were evaluated by surface plasmon resonance (SPR) using a

ProteOn™ XPR36 protein interaction array system (BioRad, Hercules, CA, USA) and compared to non-randomized ABD. Recombinant human HER3-Fc chimera (cat no. 348-RB, R&D Systems), His6-tagged human HER3 (cat no. 10201 -H08H, Sino Biological, Beijing, China), His₆-tagged murine HER3 (cat no. 51003-M08H, mHER3; Sino Biological, Beijing, China), human serum albumin and murine serum albumin (Sigma-Aldrich, St. Louis, MO, USA) were immobilized to 2000-4000 RU in separate flow cells of general layer medium (GLM) sensor chips using standard amine coupling according to the manufacturer's recommendations. One ligand surface was left blank in all experiments and all analytes were injected with flow-rates of 50 μΙ/min at 25 °C. Samples were injected for 252 or 300 s and dissociation was monitored for 600 or 1000 s. The surfaces were regenerated between injections with 10 mM NaOH (HER3) or 20 mM HCI (albumin). Each analyte was serially diluted to five different concentrations in running buffer (PBST pH 7.4) and injected with a simultaneous blank injection (PBST). All response levels were double referenced against the blank ligand channel and the buffer injection. Kinetic constants were calculated from experimental data by curve fitting to a 1 :1 Langmuir binding isotherm using the ProteOn Manager™ software version 3.1 .0.6 (BioRad). All kinetic measurements were performed at least in duplicates and human polyclonal IgG Fc (Bethyl Laboratories) and Fc-fused human ErbB2 (cat no. 1 129-ER, R&D Systems) were immobilized as negative controls in one of the experiments. Results

To investigate the binding properties of the selected ABD variants and compare them with the starting ABD polypeptide, SPR measurements of their interactions with HER3 and serum albumin from human and murine origin (hHER3, mHER3, murine serum albumin (MSA) and HSA) were performed. Kinetic data, at least duplicate experiments for each interaction, on seven selected variants (SEQ ID NO:161 -167) and the original ABD polypeptide (SEQ ID NO:335) are summarized in Table 1 below. The binding to HER3 was generally characterized by a fast on-rate, whereas the off-rate varied between the different variants (Table 1 ). ABD3-3 (SEQ ID NO:161 ) and ABD3-27 featured somewhat slower off-rates (Figure 3A), and thereby had the lowest K_D-values (10 and 12 nM, respectively) among the candidates (Table 1 ). As expected, data from immobilized Fc-hHER3 and His6-hHER3 correlated well and therefore data from both versions of the receptor are presented together. In addition, the affinities for mHER3 compared to hHER3 were almost identical (Table 1 ), which suggests a high degree of species conservation of the epitope on HER3. As expected, no binding of original ABD to HER3 was detected, whereas all phage-selected variants bound both human and mouse HER3 with affinities ranging from 10 nM to 100 nM. None of the HER3 binding ABD variants gave any signal on control surfaces immobilized with polyclonal human IgG-Fc or the related receptor hErbB2 (also known as hHER2).

Affinities to HSA were in the sub- or low nanomolar range for all variants except ABD3-18 (SEQ ID NO:163), which had a comparably modest affinity of around 100 nM (Table 1 ). As seen in Figure 3A, the analyzed HER3 binding ABD variants had a slow dissociation from HSA with off-rates (k_d) of around 10^"4 s^~1. For all analyzed ABD variants, the affinity for MSA was approximately 10-fold lower than for HSA, mainly due to faster dissociation. This correlates to what has been shown previously for G148-GA3 (Johansson et al, The Journal of biological chemistry 277 (10):81 14-8120 (2002)). Table 1: Affinity and T_m measurements for selected ABD variants hHER3

ABD

SEQ ID NO: k_a (A/TV) k_d (s-¹) K_D (nM) variant

ABD3-1 165 7.5 (±0.1)x10^b 5.6 (±0.2) x10^"2 75 [4]

ABD3-2 166 1.0 (±0.2) x10⁶ 2.7 (±0.2) x10^"2 26 [4]

ABD3-3 161 7.8 (±0.5) x10⁵ 7.6 (±0.6) x10^"3 10 [4]

ABD3-13 167 6.1 (±0.5) x10⁵ 2.4 (±0.1) x10^"2 39 [4]

ABD3-18 163 2.0 (±0.2) x10⁵ 1.8 (±0.1) x10^"2 94 [4]

ABD3-20 164 2.7 (±0.3) x10⁵ 1.6 (±0.1) x10^"2 60 [4]

ABD3-27 162 6.2 (±0.1) x10⁵ 7.4 (±2.0) x10^"2 12 [4]

ABD^* 335 - - - mHER3

ABD

SEQ ID NO: k_a (A/TV) k_d (s-¹) K_D (nM) variant

ABD3-1 165 9.1 (±0.3)x10^b 6.3 (±0.2) x10^"2 69 [2]

ABD3-2 166 9.3 (±0.2) x10⁵ 3.1 (±0.03) x10^"2 34 [2]

ABD3-3 161 8.3 (±0.2) x10⁵ 9.4 (±0.04) x10^"3 11 [2]

ABD3-13 167 4.9 (±0.01 )x10⁵ 1.8 (±0.1) x10^"2 37 [2]

ABD3-18 163 1.8 (±0.02) x10⁵ 1.8 (±0.1) x10^"2 97 [2]

ABD3-20 164 2.0 (±0.1) x10⁵ 1.7 (±0.2) x10^"2 86 [2]

ABD3-27 162 2.4 (±0.6) x10⁶ 8.6 (±3.3) x10^"3 4 [2]

ABD^* 335 - -

HSA

ABD

SEQ ID NO: k_a (MV¹) k_d (s-¹) K_D (nM) variant

ABD3-1 165 4.5 (±1.7)x10^b 5.7 (±1.8)x10^"b 0.1 [6]

ABD3-2 166 6.6 (±3.1) x10⁵ 1.7 (±0.3) x10^"4 0.3 [6]

ABD3-3 161 2.7 (±0.4) x10⁵ 1.1 (±0.3) x10^"4 0.5 [6]

ABD3-13 167 2.2 (±0.4) x10⁵ 1.8 (±0.6) x10^"4 0.8 [6]

ABD3-18 163 1.3 (±0.2) x10⁵ 1.1 (±0.3) x10^"2 85 [6]

ABD3-20 164 1.6 (±0.1) x10⁵ 2.7 (±0.2) x10^"4 1 -7 [6]

ABD3-27 162 9.2 (±0.5) x10⁴ 1.7 (±0.3) x10^"4 1.8 [3]

ABD^* 335 7.1 (±0.7) x10⁴ 5.8 (±0.5) x10^"4 8.2 [9]

Affinities were determined by SPR analysis from multiple experiments. Constants are represented as mean (±SD) from the number of interactions indicated in brackets. K_D -values were calculated by k_d / k_a.

Example 4

Biosensor analysis of HER3 binding in the presence of albumin

Materials and methods

Binding of four selected ABD variants (ABD3-3 (SEQ ID NO:161 ), ABD3-27 (SEQ ID NO:162), ABD3-20 (SEQ ID NO:163) and ABD3-18 (SEQ ID NO:164)) and the negative control ABD (SEQ ID NO:335) to HER3 in the presence of albumin was measured in a biosensor assay using a ProteOn™ XPR36 protein interaction array system to (i) assess simultaneous binding capacity and (ii) monitor binding behavior in increasing concentrations of albumin. Human (cat no. 348-RB, R&D Systems, Fc-fused,) and murine (cat no. 51003-M08H, Sino Biological, His6-tagged,) HER3 were immobilized to 1000-4000 RU on separate surfaces on a GLM-chip. 200 or 500 nM of the ABD variants was incubated with varying concentrations of HSA (0-2000 nM) before being injected over the chip for 200 s (50 μΙ/min at 25 °C). Dissociation was subsequently measured during 600 s. Differences between responses measured during association and dissociation were normalized against corresponding values obtained from reference injections without albumin. The same experiment was also performed with the ABD variants pre-incubated with MSA prior to injection over immobilized mHER3. Results

In the absence of simultaneous binding, strong competition from albumin could be anticipated considering the relative affinities for albumin and HER3 (Table 1 ). If this is the case, a higher affinity to albumin would favor the ABD:albumin interaction over binding to HER3. To investigate if the HER3- specific ABD variants were able to simultaneously bind albumin, a biosensor experiment was performed. A sandwich assay was set up where the ABD variants were pre-incubated with HSA before being injected over sensor chip surfaces with immobilized hHER3. If simultaneous binding would occur, the signal from channels without albumin would give lower responses compared to channels where the ABD variants were incubated with, and bound to, albumin before injection. No simultaneous binding could be detected for any candidate pre-incubated with an equal concentration of albumin. Instead, HER3 binding signal was lost when albumin was present.

To further look into the feasibility of the ABD variants in a potential therapeutic application, their ability to bind HER3 in the presence of increasing concentrations of albumin was assessed in a separate biosensor experiment. By incubating the ABD variants with increasing HSA

concentrations while the ABD concentration was kept constant (at 500 nM or 200 nM in two experiments) and thereafter detecting the binding to HER3 by simultaneous injections over a biosensor surface with immobilized HER3 (Figure 4A and 4B). Varying effects of competing albumin were observed for the analyzed variants and higher binding signals were obtained when 500 nM of the ABD variants and low concentrations of albumin were used (Figure 4A) compared to 200 nM with high concentrations of albumin (Figure 4B). ABD3- 18, ABD3-20 (SEQ ID NO:164) and ABD3-27 gradually lost their ability to bind to hHER3 when the concentration of albumin was increased. However, the excess of albumin required to achieve this effect was different for each variant. ABD3-27, for example, lost the ability to bind hHER3 with HSA present at a 1 :1 molar ratio. Interestingly, ABD3-3, which has similar kinetic properties as ABD3-27 for HER3 but binds albumin with higher affinity (Table 1 ), shows a significant HER3 binding even in the presence of a 10-fold molar excess of albumin (Figure 4B). The same patterns were observed when the ABD variants were incubated with MSA prior to injection over immobilized mHER3 for all candidates. Example 5

Heregulin competition

Materials and metods

Binding competition between heregulin (NRG-βΙ ) and the four ABD variants studied in Example 4 (SEQ ID NO:161 -164) for human HER3 (hHER3) was monitored by SPR using a ProteOn™ XPR36 protein interaction array system. NRG-βΙ ECD (cat no.396-HB, R&D Systems) or NRG-βΙ EGF domain (cat no. 377-HB, R&D Systems) were immobilized by amine coupling to 1000-2000 RU on a GLM-chip. HER3 (cat no. 348-RB, R&D Systems, Fc-chimera) was pre-incubated with varying concentrations of ABD variants (12.5-200 nM with 5 nM HER3) for 1 h at room temperature in PBST before being injected (50 μΙ/min at 25 °C) over the chip for 200 s. Surfaces were regenerated with 10 mM NaOH. All response levels were double referenced as described above and response levels towards the end of injection (at 160 s) were extracted. Responses from the pre-incubated samples were normalized against reference injections with only HER3. As a negative control, the original ABD polypeptide was included in the

experiment. Positive controls consisted of the NRG-βΙ EGF domain that competed with itself and of the HER3 binding variant of protein Z denoted Z05417 (SEQ ID NO:336; see e.g. WO201 1/056124) that is known to bind to the same site on HER3 as NRG-βΙ (Kronqvist et al., PEDS 24 (4)385-396 (201 1 )). Results

To investigate a potential influence of the selected binders on receptor function, a biosensor assay was employed to measure the effect of four representative HER3 binding ABD variants (SEQ ID NO:161 -164), including the strongest binders ABD3-3 and ABD3-27, on the interaction between soluble HER3 and immobilized NRG-βΙ . A constant concentration (5 nM) of HER3 was incubated with a series of different concentrations of an ABD variant. The mixtures were incubated for 1 h to reach equilibrium, which would theoretically take between 15-30 min calculated from the affinities using the method described by Hulme and Trevethick (Hulme and Trevethic, British Journal of Pharmacology 161 (6):1219-1237 (2010)), prior to injection over NRG-βΙ immobilized on a sensor chip. If the ABD variant binds to a surface that overlaps with the binding site of NRG-βΙ , a concentration dependent decrease in measured signal was expected. To compensate for variations in immobilization level, reference injections with HER3 alone were used to normalize signals obtained in all other injections. The observed binding of HER3 to the chip surface was found to decrease for all tested HER3 binding ABD variants (Figure 5) whereas no influence on binding was observed for the negative control ABD, which does not have any measurable affinity for HER3 (Table 1 ). This inhibitory effect correlates well to the affinities to HER3 of the evaluated ABD variants (Table 1 ). These data clearly demonstrate that all analyzed ABD variants, which have similar sequences but still represent somewhat different sequence clusters), have epitopes on HER3 that overlap with the binding-site for NRG-βΙ . These results were confirmed by performing the assay also with immobilized NRG-βΙ EGF using the same panel of binders.

Example 6

Flow-cvtometric evaluation of HER3 binding on cells

Materials and methods

To test for binding to HER3-expressing cells by flow cytometry, four of the selected HER3 binding ABD variants and the control protein ABD were genetically fused as monomers (ABD-Z) and homodimers ((ABD)₂-Z) with a C-terminal IgG-binding Z protein (derived from domain B of Protein A as described by Nilsson et al, Protein Eng 1 (2):107-1 13 (1987)). The Z domain was introduced to facilitate detection of bound ABD, and dimerization was performed to increase the apparent affinity and thereby the signals obtained in the flow cytometer. The constructs were assembled from PCR products using specific primers and sub-cloned into the same expression vector as described in Example 1 , using restriction sites EcoRI and Ascl (NEB). ABD variant and Z domain were expressed in fusion using a G(S3G)2-linker introduced by primers with a BamHI (NEB) restriction site. All proteins were expressed in Rosetta (DE3) E. coli cells, purified on HSA-Sepharose followed by an additional purification on IgG-Sepharose using the same protocol described above for both purification steps. IgG affinity purification was included to (i) achieve a higher homogeneity and (ii) verify the functionality of the Z domain in the fusion proteins. The proteins were characterized by SDS- PAGE and mass spectrometry, and binding to HER3 was confirmed by SPR as described in Example 3. Human HER3-expressing AU565 cells (American type culture collection, ATCC) were cultured to 80-90 % confluence in RPMI1640 medium (Sigma Aldrich) supplemented with 10 % fetal bovine serum (FBS) (Sigma Aldrich). As a control, SKOV-3 cells (HER3^|0W; ATCC) cultured in McCoy's 5A modified medium (Sigma Aldrich), were also treated and analyzed in the same manner. Cells were harvested by incubation in PBS supplemented with 5 mM EDTA and 0.1 mg/ml trypsin (Sigma Aldrich) for 5 min at 37 °C and, following centrifugation, re-suspended in fresh medium. 2 x 10⁵ - 7 x 10⁵ cells were incubated with 500 nM ABD-Z at room

temperature for 1 h. Binding to the cell surface was detected through incubation with biotinylated polyclonal human IgG, followed by incubation with streptavidin R-phycoerythrin conjugate (SAPE; Invitrogen). The cells were washed with 3 % PBSTB between incubations and prior to flow-cytometric analysis. The median fluorescence intensities of the analyzed cells were compared to the auto-fluorescence of non-labeled cells and cells incubated with detection reagents only (background). Non-randomized ABD, fused to Z as a monomer or dimer as described above, was used as a negative control and all analyses were performed at least in duplicates.

To verify binding specificity to HER3 on AU565, the binding of ABD variants to cells was blocked in separate experiments with NRG-βΙ EGF (cat no. 396-HB, R&D Systems). 2 x 10⁵ - 7 x 10⁵ cells were incubated with 50 nM NRG-βΙ EGF for 30 min at room temperature followed by incubation with 500 nM ABD-Z or (ABD)₂-Z for 30 min. Bound ABD variants were detected using biotinylated human IgG and SAPE as described above. The cells were washed with 3 % PBSTB between each incubation step and prior to flow- cytometric analysis. All flow-cytometric data was analyzed using Kaluza flow analysis software version 1 .0 (Beckman Coulter, Brea, Ca, USA).

Results

Binding of the four selected ABD variants tested above to the HER3- expressing human breast cancer cell-line AU565 was analyzed by flow- cytometry. Mono- or dimeric ABD variants fused to a C-terminal Z domain were incubated with the cells and bound ABD variants were detected through biotinylated IgG and a streptavidin R-phycoerythrin conjugate. ABD3-3 and ABD3-27 bound strongly to the AU565 cells, whereas no binding was observed for the ABD control, here denoted ABD^* (Figure 6A). Only weak binding signals just above background were observed for ABD3-18 or ABD3- 20 in this assay. The signal from monomeric ABD3-3 compared to ABD3-27 was lower than expected from the comparable affinities for HER3 of those ABD variants. As expected, no binding could be detected for either variant to the low HER3-expressing cell-line SKOV-3. Similar observations were made for the corresponding bivalent constructs ((ABD)₂-Z, where the (ABD)₂-Z variants of ABD3-3 and ABD3-27 gave even stronger signals than the corresponding ABD-Z molecules (Figure 6A), however unspecific binding of (ABD3-27)₂-Z was detected to the low HER3-expressing cell-line SKOV-3. The dimeric ABD3-18 and ABD3-20 did not bind the cells.

To better understand those results, the dimers were evaluated in an

SPR experiment. Dimers of ABD3-3 and ABD3-27 displayed slower off-rates, as a result of avidity, when injected over a surface immobilized with hHER3 (data not shown). In the same experiment, it was confirmed that fusion of a C- terminal Z-domain to ABD3-18 or ABD3-20, both in the form of mono- and dimers, resulted in weakened binding to HER3. This explains the lack of binding to the cells.

The specificity of ABD3-3 and ABD3-27 was further assessed on AU565-cells by pre-incubating the cells with NRG-βΙ EGF. The cells were subsequently incubated with 500 nM of ABD variant fused to a C-terminal Z- domain and detected as described above. The fluorescence intensity measured when cells had been pre-incubated with NRG- β1 was close to or equal to the background intensity (Figure 6B). Both monomeric och dimeric forms of ABD3-3 and ABD3-27 were thus blocked from binding to HER3 on the cell surface.

Summary of Examples 7-8

The inventors continue to design an affinity maturation library, and perform three rounds of flow-cytometric sorting selection from this library to identify second generation HER3 binding polypeptides which exhibit binding affinity for both HER3 and albumin. The selection is performed twice, yielding very similar results. Example 7

Library design and preparation of library vector

Materials and metods

Next, an affinity maturation library (Figure 7), was designed based on the characteristics of the ABD variants identified in the selection described in Example 1 . The resulting theoretical library size was 1 .8 x 10⁷ and 4.4 x 10⁶ variants on the nucleotide and protein level, respectively. The library was produced as a 189 bp degenerate oligonucleotide (Integrated DNA

Technologies, San Diego, CA, USA) encoding the full ABD variant library, a leading region as well as Xhol and Nhel flanking restriction sites.

This library oligonucleotide was amplified by PCR, and the resulting library insert was purified with QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). The staphylococcal display vector pSCABDI (Nilvebrant et al, PLoS ONE 6:e25791 (201 1 )) was amplified by transformation into E. coli RRIAM15 (Riitger et al, Nucleic Acids Res 10:5765-5772 (2000)), grown overnight at 37 °C in tryptic soy broth (TSB; Merck, Darmstadt, Germany) supplemented with 100 g/ml ampicillin (Sigma-Aldrich, St. Louis, MO, USA), and purified using a Jetstar Maxi Kit (Genomed, Bad Oeynhausen, Germany). The vector and library insert were separately restricted (Xhol and Nhel; New England Biolabs (NEB), MA, USA) at 37 °C overnight. Following preparative gel electrophoresis on a 2 % agarose gel, the vector and library insert were purified by QIAquick Gel Extraction kit (Qiagen). Restriction and purification were repeated for the vector but with the inclusion of a dephosphorylation step (1 h, 37 °C; Antarctic Phosphatase, NEB) before purification. The library insert was ligated to the display vector at a 5-fold molar excess using T4 DNA Ligase (NEB) at 16 °C overnight. The ligation product was purified using a QIAquick Gel Extraction kit (Qiagen) and the concentration was determined using Nanodrop spectrophotometry (Thermo Fisher Scientific, Waltham, MA, USA). The ligation product was transformed into E. coli SS320 (Sidhu et al, Methods Enzymol 328:333-363 (2000)) for amplification. The E. coli cells had been made electrocompetent through several washing steps in deionized (Dl) water and then frozen at -80 °C in 10 % glycerol. 25 successful

electroporations (2.5 kV, 5.5 ms; MicroPulser, Bio-Rad Laboratories,

Hercules, CA, USA) at 50 μΙ with ~10⁹ cells and 250 ng ligation product were performed. After 1 h recovery period (150 rpm, 37 °C) in TSB supplemented with yeast extract (TSB+Y; Merck), 20 % glucose, 10 mM MgCI₂, 10 mM MgSO₄, 10 mM NaCI and 2.5 mM KCI (SOC-medium), the number of transformed E. coli for each transformation was determined by plating onto agar plates (blood agar with NaCI, peptone and yeast extract; Merck) supplemented with 100 g/ml ampicillin. The transformed bacteria were pooled and split equally into two 5 I shake flasks with 0.5 I TSB+Y

supplemented with 100 g/ml ampicillin and grown overnight (150 rpm, 37 °C).

The overnight cultures were centrifuged (2000 ^x g, 10 min) and 10 % was re-suspended in 20 % glycerol and frozen at -80 °C. The remainder was purified using nine parallel Jetstar Maxi kits (Genomed). Two serial phenol :chloroform-extractions (Sigma-Aldrich) of the aqueous phase and subsequent ethanol precipitation were used to further purify the plasmid.

The purity and concentration of the purified plasmid were evaluated using both Nanodrop spectrophotometry (Thermo Fisher Scientific) and gel electrophoresis on 1 % agarose with MassRuler (Fermentas, Glen Burnie, MD, USA). 93 of the individual colonies on the agar plates were PCR screened and the PCR products were both examined on a 1 % agarose gel and sequenced by Sanger DNA sequencing. Sequencing was performed using Big Dye terminators (GE Healthcare, Uppsala, Sweden) and the fragments were analyzed on an ABI Prism 3700 DNA sequencer (Applied Biosystems, Foster City, CA).

Using the methodology described by Lofblom et al, (Lofblom et al, J Appl Microbiol 102:736-747 (2007)), the purified pSCABDI containing the library insert was transformed into Staphylococcus carnosus (S. carnosus). S. carnosus TM300 (Augustin and Gotz, FEMS Microbiol. Lett. 54:203-207

(1990)) were grown overnight in B2-medium (Lofblom et al, (2007) supra) and made electrocompetent through several washing and centrifugation steps (3000-5500 x g, 4 °C, 10 min) in Dl-water and subsequently frozen at -80 °C in 10 % glycerol. 212 successful electroporations (2.3 kV, 1 .1 ms) were performed in 50 μΙ (0.5 M sucrose, 10% glycerol) with ~10¹⁰ cells and 6 g plasmid each. The bacteria recovered (1 h, 37 °C, 150 rpm) in B2 medium in pooled groups of approximately ten electroporations before a sample of each pool was plated in duplicates onto agar plates supplemented with 10 g/ml chloramphenicol (cml; Sigma-Aldrich) to determine the transformation frequency. The pools were pooled pairwise, and inoculated to eleven 5 I shake flasks with 0.5 I B2 medium supplemented with 10 g/ml cml. After 20 h incubation (37 °C, 150 rpm) the pools were harvested (4000 * g, 4 °C, 8 min) and frozen at -80 °C in 15-20 % glycerol. An aliquot from each pool was thawed and plated onto agar plates supplemented with 10 g/ml cml to determine the cell density. A master stock, which represented all transformed bacteria equally, was made using the transformation frequency and cell density. This master stock was used for all subsequent analyses and sortings. 651 of the individual colonies on the agar plates were picked at random, PCR screened and sequenced to enable comparison of the actual transformed library to the theoretical design. Analysis of sequence data was performed using Geneious (Drummond et al, Geneious v.5.6, available from

http://www.geneious.com (2012)) and R (R Development Core Team, available from http://www.R-project.org (201 1 )).

Results

The display vector and randomized library insert were successfully ligated. A resulting library coverage of >12x and >3x on the protein (4.4 x 10⁶ variants) and gene (1 .8 x 10⁷ variants) level, respectively, was achieved in all steps. The achieved coverage indicates a high integrity, i.e. that the complete library was successfully transformed into S. carnosus. A sequence logotype representing the library is shown in Figure 12.

651 single colonies of transformed bacteria were sequenced to evaluate the integrity and fidelity of the transformed sequences to the library design. No dummy sequences (from the starting plasmid) were found and in almost 90% (575) of all sequences an ABD variant sequence could be identified demonstrating a low background of cells without the library plasmid (Figure 8A). The ABD variant sequences were analyzed further, and 38 % (217) were found to contain full-length sequences consistent with the design (Figure 8B). Significant fractions containing sequences with deletions (27 %) or incomplete sequences (28 %) were also observed. Only a few percent contained mutations outside the randomized sites, inserts or combinations of inserts, deletions and mutations. The full-length sequences were analyzed and the relative frequency of each amino acid at every randomized position was determined (Figure 8C). All expected amino acids were observed on every randomized position and the difference between observed values and the design in relative frequency was close to zero (no difference) for almost all randomized positions and amino acids (Figure 8D). These data

demonstrate that at least one third of all cells contained full-length sequences of ABD variants with high fidelity towards the design. Example 8

Flow-cvtometric sorting and isolation of second generation HER3 binding

ABD variants

Materials and methods

Human Hise-tagged HER3 (cat no. 10201 -H08H, Sino Biological) was dissolved in PBS (150 mM NaCI, 8 mM Na₂HPO₄, 2 mM NaH₂PO₄; pH 7.4) supplemented with 0.1 M NaHCOsto protein concentrations of 67 g/ml and 1 mg/ml, respectively. Biotinylation was performed by incubation with a 25-fold molar excess of biotin succinimidyl ester (Biotin- XX-NHS; Invitrogen,

Carlsbad, CA, USA). After 1 h at room temperature (RT), an excess of glycine was added to each mixture and the mixtures were dialyzed (10 kDa MW cutoff; Slide-A-Lyzer; Pierce Biotechnology, Rockford, IL, USA) against PBS overnight at 4 °C.

A schematic illustration of the flow cytometric analysis and the FACS- sorting is shown in Figure 9. More than a 10-fold excess, compared to the theoretical library size on the protein level, of staphylococcal cells containing the library plasmid were inoculated to 10, 100 or 500 ml TSB+Y

supplemented with 10 g/ml cml. After overnight incubation (37 °C, 150 rpm), the optical density at 578 nm (OD₅₇₈) was measured. Using the linear relationship ODs78 = 1 <→ ~10⁸ cells/ml, established by plating onto agar plates, ~10⁸ cells were added to 1 ml PBS supplemented with 0.1 % Pluronic surfactant (PBSP; BASF, Mount Olive, NJ, USA), washed (pelleted (3500xg, 6 min, RT), re-suspended in 180 μΙ PBSP and pelleted again) and re- suspended in PBSP with 50 nM biotinylated HER3 or 1/100 diluted Alexa Fluor 488 (Invitrogen) conjugated to human serum albumin. Equilibrium binding was achieved by incubation at RT with gentle mixing for 1 h, >5 times the theoretical time required for the highest-affinity first generation (phage display) binders to reach 97 % equilibrium as determined using the

methodology described by Hulme and Trevethick (Hulme and Trevethick, Br. J. Pharmacol. 161 :1219-1237 (2010)). After washing with ice-cold PBSP, the cells were re-suspended in a mixture of 1 .25 g/ml streptavidin conjugated to R-Phycoerythrin (SAPE; Invitrogen) and 10, 100 or 320 nM Alexa Fluor 647 (Invitrogen) conjugated to human polyclonal IgG in ice-cold PBSP and incubated on ice for 30 min. As staphylococcal cells can display ~10⁴ recombinant proteins on their surface (Lofblom et al, (2007) supra), the volumes used during both incubations were adjusted to achieve a molar excess of >10⁵ of ligands (HER3, HSA-488, SAPE and lgG-647) to cells, thus reducing any depletion effects that could otherwise reduce the actual concentration of available ligands. After washing, the cells were re-suspended in ice-cold PBSP and analyzed in a flow cytometer (Gallios; Beckman Coulter, Brea, CA, USA). Flow cytometric data was analyzed in FCS Express 4 (De Novo Software, Los Angeles, CA, USA). Cell debris and aggregates were excluded in the analysis by gating the main population in forward and side scatter (FSC/SSC). Surface expression levels were analyzed by measuring the binding of the fluorescent reporter (lgG-647) to the dimeric Z domain (Nilsson ef a/ (1987), supra). Around 10 times the number of transformed S. carnosus (>100 times the library size on the protein level) were inoculated to 100 ml TSB+Y supplemented with 10 g/ml cml. After overnight incubation (37 °C, 150 rpm), the optical density at 578 nm (OD₅7s) was measured and 10 times the number of transformed S. carnosus cells were added to 1 ml PBSP. Washing and incubation steps were performed as described for flow

cytometric analysis. 50 nM and 10 nM HER3 were used during the first and second round of HER3 sorting, respectively. 1 .25 g/ml SAPE and 10 nM lgG-647 were used during HER3 sortings. During the first HER3 sorting, >100 times the library size (on the protein level) of cells were interrogated and the lgG⁺-cells with the highest SAPE signal (HER3 binding) were gated and sorted directly into 1 ml TSB+Y using a fluorescence activated cell sorter (MoFlo Astrios; Beckman Coulter). After incubation for 1 h (37 °C, gentle mixing), the cells were inoculated to a final volume of 10 ml TSB+Y

supplemented with 10 g/ml cml in a 100 ml shake flask. After overnight growth (37 °C, 150 rpm), 85 % glycerol was added to a final concentration of 15-20 % and the cells were frozen at -80 °C. For the second HER3 sorting, more than 10 times the number of cells sorted during the first round were treated as described above and interrogated (-15,000 events/s). The lgG⁺ cells with the highest SAPE signals were sorted, grown overnight in 3 ml TSB+Y with cml and frozen at -80 °C, analogous to round 1 . Round 3 was performed in three variants, wherein the concentration of HER3 was varied to 0.1 nM, 1 .0 nM and 10 nM, respectively (all other experimental details for round 3 were performed essentially as described above). Cells sorted from rounds 1 , 2 and 3 were analyzed in the flow cytometer for maintained HSA binding and enriched HER3 binding (Figure 10). After the third sorting round, isolated cells were spread on agar plates and selected variants were identified by DNA sequencing.

Additionally, FACS sorting of ABD, ABD_HER3-3 and a Z variant polypeptide with a sub-nanomolar HER3 affinity (Z05417 (SEQ ID NO:336)) using 10 nM HER3 was perfornned essentially as described above (Figure 10).

During all sorting, the FACS was cooled to 6 °C to minimize

dissociation of protein from cell surfaces.

A replicate selection, completely separate from the first, was performed in order to verify that the selection system was reproducible, and in order to check whether the same top-performing clones were isolated a second time. The sort gates were set to mimic the selection conditions of the first attempt, in order to apply similar selection stringencies. Results

To identify and sort HER3 binding variants of the library, the

Staphylococci were incubated with fluorescently labeled HER3 and sorted by FACS. Three successive rounds of sorting with increasing stringency were performed (Figure 10). Retained HSA binding was also confirmed.

Enrichment of HER3 binding polypeptides was observed over the three sorting rounds. Much stronger HER3 binding signals were obtained from candidates in the affinity maturation library as compared to ABD3-3 identified as described in Example 1 , which is the first generation clone having the highest affinity. A comparison with the Z variant polypeptide Z05417, which has a sub-nanomolar HER3 affinity and was used as a positive control, indicates that the selection was successful. Interestingly, more sub- populations can be distinguished in the output from selections performed with a lower target concentration in the third round.

After three rounds of flow-cytometric sorting of the affinity maturation library, the selected variants were sequenced, which generated a set of 402 selected sequences. Of the 402, 160 sequences were unique, and are listed in Figure 1 with the sequence identifiers SEQ ID NO:1 -160. Sequence logos representing the output from the selections with 0.1 , 1 .0 or 10 nM HER3 are shown in Figure 12A, 12B and 12C, respectively. A sequence logo

representing the pooled data from these three selections is shown in Figure 14. Logotypes were generated using Weblogo 3.3 (Crooks et al, Genome Research 14:1 188-1 190 (2004)). The overall height of each residue corresponds to its degree of conservation and the height within each stack relates to the relative frequency. The maximum sequence conservation per site is described by Iog2(20) for 20 possible amino acids (^« 4.3 bits).

Nine sequences (SEQ ID NO:1 -9) were found to be significantly over- represented in the dataset and corresponded to 43.53 % of the total number of sequenced variants (Table 2 and Figure 14). In particular, variant 23_A01 (SEQ ID NO:1 ) represented almost 13 % of the sequenced variants. The said nine sequences exhibited a high level of % identity, differing only in three residue positions (Figure 14).

The binding profiles from the replicate selection yielded similar results, showing an almost identical enrichment between each round. Again, the 10 nM track in round 3 isolated the best binders. When outputs were compared during flow-cytometric analysis after selection, the cell populations had very similar characteristics. This indicated that many of the isolated clones are the same in both replicates (Figure 15).

In summary, using combinatorial protein engineering, the inventors construct and evaluate an affinity maturation library of variants based on ABD and identify polypeptides with dual specificity towards albumin and HER3. The identified HER3 binding polypeptides (SEQ ID NO:1 -167) represent novel HER3 binding molecules with the favorable properties of small size and long serum half-life.

Table 2: Statistics over the nine most common sequences from round 3 of affinity maturation

Total # of Visible in

Designation SEQ ID NO: sequences Percent of all track(s)

23 A01 1 52 12.94 % 23 C A B

24 D C08 2 33 8.21 % B A C D 23

24 D D03 3 23 5.72 % D 23 C A B

24 D G10 4 14 3.48 % 23 D C A B

23 G07 5 13 3.23 % D 23 C A B

23 D C10 6 12 2.99 % D 23 C A B

23 C06 7 1 1 2.74 % 23 C A B

23 G10 8 10 2.49 % 23 C A B

23 G1 1 9 7 1 .74 % 23 C A B

SUM 175 43.53 % Example 9

Off-rate selection of HER3 binding ABD variants using flow-cytometric sorting Summary

To narrow down the output towards the variants with the highest affinity, an off-rate selection step, in which clones with slower dissociation kinetics are favored, was performed in two parallel tracks.

Materials and methods

Cells corresponding to a 10-fold excess of the number of sorted cells in round 3 in Example 8 (10 nM track) were incubated to equilibrium with an excess of biotinylated HER3. After non-binding HER3 had been washed away, an excess of non-labeled HER3 was added to minimize re-association, and incubated for 30 min or 3 h in respective track. Flow-cytometric sorting and identification of clones by sequencing was performed as described above.

Results

Despite the harsh conditions in this fourth round, clear binding populations were visible during sorting in both tracks. Comparing the two incubation times, a clear difference in the fluorescence intensity of HER3 detection was visible, indicating that a degree of dissociation had occurred after 3 h (Figure 15). Moreover, a very low number of non-binding clones were present, as was also true when analyzing the output from round 3 described in Example 8 above. This indicated that the population of clones had similar dissociation kinetics.

Sequencing of the clones sorted during the off-rate selection showed that the most common clones were identical in both tracks, with one dominating clone. When comparing the sequence data of the off-rate selection and round 3, it became clear that a convergence toward a specific set of sequences with a high degree of similarity had occurred. The off-rate selection also identified 50 new, unique variants. These are listed in Figure 1 and in the accompanying sequence listing as SEQ ID NO:338-387. By way of deduction, the mutations conferring HER3 binding to the starting ABD sequence in these variants were also virtually introduced into a previously described ABD variant sequence, disclosed and studied in WO2012/004384, and the resulting, deduced, sequences are listed in Figure 1 and the sequence listing as SEQ ID NO:388-437.

Example 10

Flow-cytometric screening of selected second generation HER3 binding ABD variants

Summary

Flow-cytometric screening was performed to rank the selected HER3 binding variants based on their affinity for HER3. Screening of 165 of the selected, unique clones revealed that the more frequently observed clones were among those clones that exhibited the best binding signals. Materials and methods

165 unique clones, identified from the 10 nM track after three rounds of selection as described in Example 8 or from the off-rate selection described in Example 9, were grown individually in 96-deep well plates in 1 ml TSB+Y. 10⁶ cells were transferred from each culture, based on measurements of the cellular density at OD578, and washed by centrifugation (3000 g, 4 °C, 6 min) and re-suspension in PBSP. Subsequently, each clone was incubated with 5 nM biotinylated rhHER3 (Sino Biological) in PBSP for 1 h at RT. All subsequent washing and incubation steps were performed at 4 °C or on ice. Detection and expression normalization was performed by incubating with SAPE (Invitrogen) and lgG-647 for 30 min. HSA conjugated to Alexa-488 (HSA-488) was used for expression normalization of the positive control Z05417 (SEQ ID NO:336). HER3-binding was analyzed by interrogating 5 x 10⁴ cells in a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) by measuring the median fluorescence intensity (MFI) of both SAPE and IgG- 647.

Results

A set of 165 variants were selected for screening against HER3 binding. Small variations in expression levels were observed between clones and between days. Therefore, all binding signals were normalized against expression levels before comparisons were made. The screening result, including all 165 variants, is shown in Figure 16A. A repeated ranking of the 28 clones showing the highest binding signal and sequence frequency is shown in Figure 16B. Several clones among those most frequently observed during sequencing were identified as the strongest binding clones. As expected, many of the rare clones only identified once during sequencing had low binding signals, which explain their low accumulation. Clones with binding signals close to the background intensity were verified as positive HER3- binders in a separate experiment with a higher HER3 concentration (Figure 16C). Thus, it was confirmed that all 165 isolated clones were able to bind to HER3. The binders with the highest binding signals were comparable to the positive control Z05417 (SEQ ID NO:336), while the lower signals observed for the least frequent clones were comparable to that of the first generation variant ABDHERS-S (SEQ ID NO:161 ).

Example 1 1

Characterization of second generation HER3 binding ABD variants

Summary

The top five variants from the affinity screening (SEQ ID NO:9, SEQ ID NO:4, SEQ ID NO:54, SEQ ID NO:86 and SEQ ID NO:83) and, for

comparative purposes, five additional variants (SEQ ID NO:2, SEQ ID NO:1 , SEQ ID NO:150, SEQ ID NO:33 and SEQ ID NO:345) were selected for cloning, expression and further characterization by CD and SPR. Variants SEQ ID NO:2 and SEQ ID NO:1 occurred at high frequencies during sequencing, variants SEQ ID NO:150 and SEQ ID NO:33 represented the last two of the 28 best variants in the screen described in Example 9, and variant SEQ ID NO:345 had an amino acid deletion in position 43 which had not been included in the library design. Variants SEQ ID NO:150 and SEQ ID NO:33 were included in the characterization to measure the difference in affinity between the best and worst performing variants of the top 28 variants, in order to assess the sensitivity of the screening assay.

Materials and methods

Protein production and purification: Rosetta colonies were used to inoculate over-night cultures with TSB supplemented with 20 g/ml

chloramphenicol and 50 g/ml kanamycin. 1 ml culture was used the next day to inoculate 100 ml cultures with TSB+Y supplemented with 20 g/ml chloramphenicol and 50 g/ml kanamycin. At OD₆oo ^s 0.6, expression was induced by the addition of IPTG to a final concentration of 1 mM and the cultures were incubated over night at 25 °C. The next day, the cells were harvested by centrifugation (3000 g, 4 °C, 20 min) and resuspended in TST, pH 8.0. Cells were lysed by sonication using a Vibra-cell sonicator (Sonics and Materials Inc., Newtown, CT, USA) and cell debris was removed by centrifugation (10000 g, 4 °C, 20 min). Lysates were filtered through a

0.45 μιτι filter and loaded onto a column with 7.5 ml (column volume, CV) HSA sepharose equilibrated with 10 CV TST. The column was washed with 7 CV NH₄Ac, pH 5.5, and finally eluted in 1 ml fractions with HAc, pH 2.8. Fractions containing protein, as determined by absorbance measurements at 280 nm, were dried overnight in a Savant AES2010 SpeedVac system

(Thermo Scientific, Rockford, IL, USA). The proteins were re-suspended in PBS and purity was confirmed by SDS-PAGE electrophoresis and mass spectrometry using a 6520 Accurate Q-TOF LC/MS (Agilent, Santa Clara, CA, USA). The concentrations were measured in triplicates using a bicinchoninic acid kit (Sigma-Aldrich) according to the supplier's recommendation using an amino-acid analyzed ABD variant as standard (ABD_HER3-3)- Secondary structure and stability were verified by circular dichroism spectroscopy, performed as described in Example 2.

Affinity measurements: The affinities of expressed ABD variants to HER3 and albumin were assessed by surface plasmon resonance (SPR) experiments essentially as described in Example 3. In brief, HER3 (Sino Biological) and serum albumin (Sigma Aldrich) were immobilized on a general layer medium (GLM) and a general layer coupling (GLC) chip, respectively. The HER3 proteins were immobilized to approximately 2000-2500 RU and the albumin proteins to approximately 1500-2000 RU. PBST was used as running buffer and all injections were performed at 50 μΙ/min. All analyzed HER3 binding ABD variants were serially diluted in PBST to between 50 and 0.5 nM and injected with an association time of 300 s. The dissociation was monitored for 2000 s.

Results

All 10 subcloned candidates were successfully expressed and purified in high yields. A typical three-helix bundle secondary structure as well as a high thermal stability was observed for all 10 clones as determined by circular dichroism spectroscopy. Affinities to the human and murine forms of HER3 and albumin were determined by SPR analysis with good reproducibility, and the K_D values were shown to lie in the low nanomolar to sub-nanomolar range with respect to both HER3 and albumin (Table 3). The affinities to the human and murine forms of HER3 were very similar (ranging from 0.6 to 9.6 nM) for all analyzed ABD variants, while the affinity to MSA was approximately 10- fold lower than the affinity to HSA. Surprisingly, the variant SEQ ID NO:1 had a higher affinity than the variant SEQ ID NO:83, which had consistently ranked high in all screening experiments. The two clones with the lowest binding signal among the top 28 during screening exhibited a lower affinity to HER3 as expected. On the other hand, the variant SEQ ID NO:345 had a higher affinity than both of these, which does not correlate with the screening result. The results for the deletion variant SEQ ID NO:345 indicate that the last randomized position has little or no impact on binding to HER3. This is also supported by the high level of divergence in that position for all other isolated clones.

Table 3: Affinity measurements for selected second generation HER3 binding ABD variants hHER3

SEQ ID NO: k_a (MV¹) k_d (s-¹) K_D (M)

9 1.4(±0.1)x10 8.1 (±1.0) x10^"4 5.6(±0.4) x10-^1u

4 2.1 (±0.3) x10⁶ 1.2(±0.1)x10^"3 6.0(±1.6) x10^"10

2 1.8(±0.2)x10⁶ 2.5(±0.3) x10^"3 1.5(±0.3) x10^"9

1 1.7(±0.2)x10⁶ 1.3(±0.1)x10^"3 7.8(±0.9) x10^"10

150 1.9(±0.2)x10⁶ 4.3(±0.5) x10^"3 2.3(±0.4) x10^"9

54 8.1 (±0.5) x10⁵ 1.6(±0.1)x10^"3 2.0(±0.2) x10^"9

86 8.6(±1.0) x10⁵ 1.9(±0.04)x10^"3 2.3(±0.3) x10^"9

83 1.4(±0.6)x10⁶ 1.2(±0.2)x10^"3 9.3(±2.6) x10^"10

33 2.1(±0.1)x10⁵ 1.9(±0.1)x10^"3 8.9(±0.7) x10^"9

345 1.1(±0.04)x10⁶ 2.0(±1.0) x10^"3 1.8(±0.1) x10^"9 cn

ITEMIZED LISTING OF EMBODIMENTS

1 . HER3 binding polypeptide, comprising an amino acid sequence selected from: i) LAX3AKX6X7AX9X10 XiiLDXi₄Xi₅GVSDX₂o YKX₂₃LIDKAKT

VEGVX35ALX38X39X40 ILX43ALP wherein, independently of each other,

Xs s selected from D, Q, R, S and T;

Xe s selected from A, K, R and T;

X7 s selected from L, R and V;

X₉ s selected from L and N;

X10 s selected from H, R and Y;

X11 s selected from F, I, L, M and V;

Xl4 s selected from A, D, E, G, H, K, L, M, N, P, Q, R, S, T and V;

Xl5 s selected from K, R, T and V;

X20 s selected from F and Y;

X23 s selected from D and R;

X35 s selected from H, M, Q and R;

X38 s selected from A, I, L, S, T and V;

X39 s selected from F, I, L, R and S;

X40 s selected from E and A; and

X43 s selected from A, G H ,l L, P, R, T and V; and ii) an amino acid sequence which has at least 93 % identity to the sequence defined in i).

2. HER3 binding polypeptide according to item 1 , wherein X₃ in sequence i) is selected from R, S and T. 3. HER3 binding polypeptide according to item 2, wherein X₃ in sequence i) is selected from S and T.

4. HER3 binding polypeptide according to item 2, wherein X₃ in sequence i) is selected from R and T.

5. HER3 binding polypeptide according to any one of items 3-4, wherein X₃ in sequence i) is T. 6. HER3 binding polypeptide according to item 4, wherein X₃ in sequence i) is R.

7. HER3 binding polypeptide according to item 3, wherein X₃ in sequence i) is S.

8. HER3 binding polypeptide according to any preceding item, wherein Xe in sequence i) is selected from A, K and R.

9. HER3 binding polypeptide according to item 8, wherein X₆ in sequence i) is selected from K and R.

10. HER3 binding polypeptide according to item 9, wherein X₆ in sequence i) is R. 1 1 . HER3 binding polypeptide according to item 9, wherein X₆ in sequence i) is K.

12. HER3 binding polypeptide according to any preceding item, wherein X₇ in sequence i) is selected from L and R.

13. HER3 binding polypeptide according to item 12, wherein X₇ in sequence i) is L.

14. HER3 binding polypeptide according to any preceding item, wherein X₉ in sequence i) is L.

15. HER3 binding polypeptide according to any one of items 1 -13, wherein X₉ in sequence i) is N. 16. HER3 binding polypeptide according to any preceding item, wherein Xi₀ in sequence i) is selected from H and Y.

17. HER3 binding polypeptide according to item 16, wherein Xi₀ in sequence i) is Y.

18. HER3 binding polypeptide according to any one of items 1 -15, wherein Xi₀ in sequence i) is R.

19. HER3 binding polypeptide according to any preceding item, wherein X in sequence i) is selected from F, I, L and V.

20. HER3 binding polypeptide according to item 19, wherein X in sequence i) is selected from F, L and V.

21. HER3 binding polypeptide according to item 19, wherein X in sequence i) is selected from I, L and V. 22. HER3 binding polypeptide according to any one of items 20-21 , wherein X in sequence i) is selected from L and V.

23. HER3 binding polypeptide according to item 22, wherein X in sequence i) is L.

24. HER3 binding polypeptide according to any preceding item, wherein Xi in sequence i) is selected from A, D, G, K, L, N, P, Q, R, S and T.

25. HER3 binding polypeptide according to item 24, wherein Xi in sequence i) is selected from A, D, G, K, L, N, P, Q, R and S.

26. HER3 binding polypeptide according to item 25, wherein Xi in sequence i) is selected from D, G, K, Q, R and S.

27. HER3 binding polypeptide according to item 26, wherein Xi in sequence i) is selected from D, G, K, R and S. 28. HER3 binding polypeptide according to item 26, wherein Xi in sequence i) is selected from G, K, Q, R and S.

29. HER3 binding polypeptide according to item 27, wherein Xi in sequence i) is selected from D, G, R and S.

30. HER3 binding polypeptide according to any one of items 27-28, wherein Xi in sequence i) is selected from G, K, R, and S. 31 . HER3 binding polypeptide according to item 30, wherein Xi in sequence i) is selected from G, K and R.

32. HER3 binding polypeptide according to item 31 , wherein Xi in sequence i) is selected from G and R.

33. HER3 binding polypeptide according to item 31 , wherein Xi₄ in sequence i) is selected from G and K.

34. HER3 binding polypeptide according to item 30, wherein Xi₄ in sequence i) is selected from S and R.

35. HER3 binding polypeptide according to any one of items 32-33, wherein Xi in sequence i) is G. 36. HER3 binding polypeptide according to any one of items 32 and

34, wherein Xi₄ in sequence i) is R.

37. HER3 binding polypeptide according to item 33, wherein Xi₄ in sequence i) is K.

38. HER3 binding polypeptide according to item 34, wherein Xi in sequence i) is S.

39. HER3 binding polypeptide according to any preceding item, wherein Xi₅ in sequence i) is selected from R, T and V. 40. HER3 binding polypeptide according to item 39, wherein Xi₅ in sequence i) is selected from R and T.

41 . HER3 binding polypeptide according to item 39, wherein Xi₅ in sequence i) is selected from R and V.

42. HER3 binding polypeptide according to any one of items 1 -38, wherein Xi₅ in sequence i) is selected from K and R. 43. HER3 binding polypeptide according to any one of items 40-42, wherein Xi₅ in sequence i) is R.

44. HER3 binding polypeptide according to item 40, wherein Xi₅ in sequence i) is T.

45. HER3 binding polypeptide according to item 41 , wherein Xi₅ in sequence i) is V.

46. HER3 binding polypeptide according to item 42, wherein Xi₅ in sequence i) is K.

47. HER3 binding polypeptide according to any preceding item, wherein X₂o in sequence i) is F. 48. HER3 binding polypeptide according to any one of items 1 -46, wherein X₂o in sequence i) is Y.

49. HER3 binding polypeptide according to any preceding item, wherein X₂3 in sequence i) is D.

50. HER3 binding polypeptide according to any one of items 1 -48, wherein X₂3 in sequence i) is R.

51 . HER3 binding polypeptide according to any preceding item, wherein X₃₅ in sequence i) is selected from H, Q and R. 52. HER3 binding polypeptide according to item 52, wherein X₃₅ in sequence i) is selected from Q and R.

53. HER3 binding polypeptide according to any one of items 1 -50, wherein X₃₅ in sequence i) is selected from H, M and R.

54. HER3 binding polypeptide according to any one of items 1 -50, wherein X₃₅ in sequence i) is selected from H, M and Q. 55. HER3 binding polypeptide according to any one of items 53-54, wherein X₃₅ in sequence i) is selected from H and M.

56. HER3 binding polypeptide according to any one of items 51 and 55, wherein X₃₅ in sequence i) is H.

57. HER3 binding polypeptide according to item 55, wherein X₃₅ in sequence i) is M.

58. HER3 binding polypeptide according to any one of items 52-53, wherein X₃₅ in sequence i) is R.

59. HER3 binding polypeptide according to any one of items 52 and 54, wherein X₃₅ in sequence i) is Q. 60. HER3 binding polypeptide according to any preceding item, wherein X₃₈ in sequence i) is selected from I, L, T and V.

61 . HER3 binding polypeptide according to item 60, wherein X₃₈ in sequence i) is selected from I, V and L.

62. HER3 binding polypeptide according to item 61 , wherein X₃₈ in sequence i) is selected from I and V.

63. HER3 binding polypeptide according to item 61 , wherein X₃₈ in sequence i) is selected from I and L. 64. HER3 binding polypeptide according to item 61 , wherein X₃₈ in sequence i) is selected from L and V.

65. HER3 binding polypeptide according to any one of items 62-63, wherein X₃₈ in sequence i) is I.

66. HER3 binding polypeptide according to any one of items 63-64, wherein X₃₈ in sequence i) is L. 67. HER3 binding polypeptide according to any one of items 1 -59, wherein X₃₈ in sequence i) is selected from A, S, T and V.

68. HER3 binding polypeptide according to item 67, wherein X₃₈ in sequence i) is selected from S, T and V.

69. HER3 binding polypeptide according to any one of items 62, 64 and 68, wherein X₃₈ in sequence i) is V.

70. HER3 binding polypeptide according to item 68, wherein X₃₈ in sequence i) is selected from S and T.

71 . HER3 binding polypeptide according to item 70, wherein X₃₈ in sequence i) is S. 72. HER3 binding polypeptide according to item 70, wherein X₃₈ in sequence i) is T.

73. HER3 binding polypeptide according to any preceding item, wherein X₃₉ is selected from F, I, L and R.

74. HER3 binding polypeptide according to item 73, wherein X₃₉ in sequence i) is selected from F, L and R.

75. HER3 binding polypeptide according to item 74, wherein X₃₉ in sequence i) is selected from L and R. 76. HER3 binding polypeptide according to item 75, wherein X₃₉ in sequence i) is L.

77. HER3 binding polypeptide according to item 75, wherein X₃₉ in sequence i) is R.

78. HER3 binding polypeptide according to any preceding item, wherein X40 in sequence i) is E. 79. HER3 binding polypeptide according to any one of items 1 -77, wherein X40 in sequence i) is A.

80. HER3 binding polypeptide according to any preceding item, wherein X43 in sequence i) is selected from A, G, H, I, P, R, T and V.

81 . HER3 binding polypeptide according to item 80, wherein X43 in sequence i) is selected from A, G, H, I, P, R and V.

82. HER3 binding polypeptide according to item 81 , wherein X43 in sequence i) is selected from A, G, R and V.

83. HER3 binding polypeptide according to item 82, wherein X43 in sequence i) is selected from G, R and V. 84. HER3 binding polypeptide according to item 83, wherein X43 in sequence i) is G.

85. HER3 binding polypeptide according to item 83, wherein X43 in sequence i) is V.

86. HER3 binding polypeptide according to item 83, wherein X43 in sequence i) is R.

87. HER3 binding polypeptide according to item 81 , wherein X43 in sequence i) is selected from A, I, H and P. 88. HER3 binding polypeptide according to item 87, wherein X43 in sequence i) is selected from A, I and H.

89. HER3 binding polypeptide according to item 88, wherein X43 in sequence i) is selected from A and I.

90. HER3 binding polypeptide according to item 89, wherein X43 in sequence i) is A.

91 . HER3 binding polypeptide according to item 89, wherein X43 in sequence i) is I.

92. HER3 binding polypeptide according to item 88, wherein X43 in sequence i) is H.

93. HER3 binding polypeptide according to any preceding item, wherein sequence i) is

LATAKX₆LAX₉Y LLDRRGVSDX₂₀ YKX23LIDKAKT VEGVQALX₃₈RX₄o

wherein, independently of each other,

Xe is selected from K and R;

Xg is selected from L and N;

X20 is selected from F and Y;

X23 is selected from D and R;

X38 is selected from I, L and V;

X40 is selected from A and E; and

X43 is selected from G, I, R and V.

94. HER3 binding polypeptide according to any one of the preceding items, wherein sequence i) is selected from SEQ ID NO:1 -334 and 338-437, such as selected from the group consisting of SEQ ID NO:1 -334. 95. HER3 binding polypeptide according to item 94, wherein said sequence i) is selected from SEQ ID NO:1 -167 and 338-387, such as selected from SEQ ID NO:1 -167. 96. HER3 binding polypeptide according to item 95, wherein sequence i) is selected from SEQ ID NO:1 -160.

97. HER3 binding polypeptide according to item 96, wherein sequence i) is selected from SEQ ID NO:1 -9.

98. HER3 binding polypeptide according to item 95, wherein sequence i) is selected from SEQ ID NO:161 -167.

99. HER3 binding polypeptide according to item 98, wherein sequence i) is selected from SEQ ID NO:161 -164.

100. HER3 binding polypeptide according to item 99, wherein sequence i) is selected from SEQ ID NO:161 -162. 101 . HER3 binding polypeptide according to item 100, wherein sequence i) is SEQ ID NO:161 .

102. HER3 binding polypeptide according to item 96, wherein sequence i) is selected from SEQ ID NO:1 -9, 54, 83 and 86.

103. HER3 binding polypeptide according to item 102, wherein sequence i) is selected from SEQ ID NO:4, 9, 54, 83 and 86.

104. HER3 binding polypeptide according to item 94, wherein said sequence i) is selected from SEQ ID NO:168-334 and 388-437, such as selected from SEQ ID NO:168-334.

105. HER3 binding polypeptide according to item 104, wherein sequence i) is selected from SEQ ID NO:168-327.

106. HER3 binding polypeptide according to item 105, wherein sequence i) is selected from SEQ ID NO:168-176. 107. HER3 binding polypeptide according to item 104, wherein sequence i) is selected from SEQ ID NO:328-334.

108. HER3 binding polypeptide according to item 107, wherein sequence i) is selected from SEQ ID NO:328-331 .

109. HER3 binding polypeptide according to item 108, wherein sequence i) is selected from SEQ ID NO:328-329.

1 10. HER3 binding polypeptide according to item 109, wherein sequence i) is SEQ ID NO:328.

1 1 1 . HER3 binding polypeptide according to item 105, wherein sequence i) is selected from SEQ ID NO:168-176, 221 , 250 and 253.

1 12. HER3 binding polypeptide according to item 1 1 1 , wherein sequence i) is selected from SEQ ID NO:171 , 176, 221 , 250 and 253. 1 13. HER3 binding polypeptide according to any preceding item, which is capable of binding to HER3 such that the K_D value of the interaction with HER3 is at most 1 x 10^"8 M, such as at most 1 x 10^"9 M, such as at most 1 x 10^"10 M, such as at most 1 x 10^{"1 1} M. 1 14. HER3 binding polypeptide according to any preceding item, which is capable of binding albumin.

1 15. HER3 binding polypeptide according to item 1 14, wherein the HER3 binding polypeptide is capable of binding to albumin such that the K_D value of the interaction with albumin is at least 1 x 10^"8 M, such as at least 1 x 10^"7 M, such as at least 1 x 10^"6 M, such as at least 1 x 10^"5 M.

1 16. HER3 binding polypeptide according to any one of items 1 14-1 15, wherein said albumin is human serum albumin.

1 17. HER3 binding polypeptide according to any preceding item as a multimer, such as a dimer. 1 18. Fusion protein or conjugate comprising

a first moiety comprising of a HER3 binding polypeptide according to any preceding item; and

- a second moiety comprising of a polypeptide having a desired biological activity.

1 19. Fusion protein or conjugate according to item 1 18, wherein said desired biological activity is a therapeutic activity.

120. Fusion protein or conjugate according to item 1 18, wherein said desired biological activity is a binding activity.

121 . Fusion protein or conjugate according to item 1 18, wherein said desired biological activity is an enzymatic activity.

122. Fusion protein or conjugate according to item 1 18 or 1 19, wherein the second moiety having a desired biological activity is a therapeutically active polypeptide.

123. Fusion protein or conjugate according to any one of items 1 18, 1 19 and 122, wherein the second moiety having a desired biological activity is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.

124. HER3 binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a cytotoxic agent.

125. HER3 binding polypeptide, fusion protein or conjugate according to item 124, wherein said cytotoxic agent is selected from the group consisting of auristatin, anthracycline, calicheamycin, combretastatin, doxorubicin, duocarmycin, the CC-1065 anti-tumorantibiotic, ecteinsascidin, geldanamycin, maytansinoid, methotrexate, mycotoxin, ricin and its analgoues, taxol and derivates thereof and combinations thereof.

126. HER3 binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a label. 127. HER3 binding polypeptide, fusion protein or conjugate according to item 126, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.

128. HER3 binding polypeptide, fusion protein or conjugate according to any preceding item, comprising a chelating environment provided by a polyaminopolycarboxylate chelator conjugated to the HER3 binding polypeptide via a thiol group of a cysteine residue or an amine group of a lysine residue.

129. HER3 binding polypeptide, fusion protein or conjugate according to item 128, wherein the polyaminopolycarboxylate chelator is 1 ,4,7,10- tetraazacyclododecane-1 , 4, 7,10-tetraacetic acid or a derivative thereof.

130. HER3 binding polypeptide, fusion protein or conjugate according to item 129, wherein the 1 ,4,7,10-tetraazacyclododecane-1 ,4,7,10-tetraacetic acid derivative is 1 ,4,7,10-tetraazacyclododecane-1 ,4,7-tris-acetic acid-10- maleimidoethylacetamide.

131 . HER3 binding polypeptide, fusion protein or conjugate according to item 128, wherein the the polyaminopolycarboxylate chelator is 1 ,4,7- triazacyclononane-1 ,4,7-triacetic acid or a derivative thereof.

132. HER3 binding polypeptide, fusion protein or conjugate according to item 128, wherein the polyaminopolycarboxylate chelator is

diethylenetriaminepentaacetic acid or derivatives thereof.

133. Polynucleotide encoding a HER3 binding polypeptide or a fusion protein according to any one of items 1 -123.

134. Method of producing a polypeptide according to any one of items 1 -123, comprising expressing a polynucleotide according to item 133. 135. Expression vector comprising a polynucleotide according to item

136. Host cell comprising an expression vector according to item 135.

137. Method of producing a polypeptide according to any one of items 1 -123, comprising

culturing a host cell according to item 136 under conditions permissive of expression of said polypeptide from its expression vector, and isolating said polypeptide.

138. Composition comprising a HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1 -132 and at least one pharmaceutically acceptable excipient or carrier.

139. Composition according to item 138, further comprising at least one additional active agent.

140. HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1 -132 or composition according to any one of items 138-

139 for use as a medicament.

141 . HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1 -132 or composition according to any one of items 138- 139 for use in diagnosis.

142. HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1 -132 or composition according to any one of items 138- 139 for use in the treatment or diagnosis of a HER3 related condition.

143. HER3 binding polypeptide, fusion protein, conjugate or composition for use according to item 142, wherein said HER3 related condition is cancer.

144. HER3 binding polypeptide, fusion protein, conjugate or composition for use according to item 143, wherein said cancer is selected from the group consisting of lung cancer, breast cancer, gastric cancer, stomach cancer, colon cancer, colorectal cancer, cancer of the small instestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, melanomas, cancers of the head and neck, pediatric gliomas, pediatric glioblastomas and astrocytomas.

145. Method of treatment of a HER3 related condition, comprising administering to a subject in need thereof an effective amount of a HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1 -132 or a composition according to any one of items 138-139.

146. Method according to item 145, wherein said HER3 binding polypeptide, fusion protein, conjugate or composition inhibits HER3 mediated signaling by binding to HER3 expressed on a cell surface.

147. Method according to any one of items 145-146, wherein said HER3 related condition is cancer.

148. Method according to item 147, wherein said cancer is selected from the group consisting of lung cancer, breast cancer, gastric cancer, stomach cancer, colon cancer, colorectal cancer, cancer of the small instestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, melanomas, cancers of the head and neck, pediatric gliomas, pediatric glioblastomas and astrocytomas.

Claims

1 . HER3 binding polypeptide, comprising an amino acid sequence selected from: i) LAX3AKX6X7AX9X10 Xi i LDXi₄X₁₅GVSDX₂o YKX₂₃LIDKAKT

VEGVX35ALX38X39X40 ILX43ALP wherein, independently of each other,

X3 is selected from D, Q, R, S and T;

Χβ is selected from A, K, R and T;

X₇ is selected from L, R and V;

X9 is selected from L and N;

X10 is selected from H, R and Y;

X11 is selected from F, I, L, M and V;

Xi₄ is selected from A, D, E, G, H, K, L, M, N, P, Q, R, S, T and V;

Xi5 is selected from K, R, T and V;

X20 is selected from F and Y;

X23 is selected from D and R;

X35 is selected from H, M, Q and R;

X38 is selected from A, I, L, S, T and V;

X39 is selected from F, I, L, R and S;

X40 is selected from E and A; and

X₄₃ is selected from A, G H ,l L, P, R, T and V; and ii) an amino acid sequence which has at least 93 % identity to the sequence defined in i).

2. HER3 binding polypeptide according to claim 1 , wherein sequence i) is

LATAKX₆LAX₉Y LLDRRGVSDX₂₀ YKX₂₃LIDKAKT VEGVQALX₃₈RX₄o

wherein, independently of each other,

Xe is selected from K and R;

Xg is selected from L and N;

X20 is selected from F and Y;

X23 is selected from D and R;

X38 is selected from I, L and V;

X40 is selected from A and E; and

X43 is selected from G, I, R and V.

3. HER3 binding polypeptide according to any one of the preceding claims, wherein sequence i) is selected from SEQ ID NO:1 -334, for example selected from SEQ ID NO:1 -167, for example selected from SEQ ID NO:1 - 160, for example selected from SEQ ID NO:1 -9.

4. HER3 binding polypeptide according to claim 3, wherein sequence i) is selected from SEQ ID NO:161 -167, for example selected from SEQ ID NO:161 -164, for example selected from SEQ ID NO:161 -162, for example SEQ ID NO:161 .

5. HER3 binding polypeptide according to claim 3, wherein said sequence i) is selected from SEQ ID NO:168-334, for example selected from SEQ ID NO:168-327, for example selected from SEQ ID NO:168-176.

6. HER3 binding polypeptide according to claim 5, wherein sequence i) is selected from SEQ ID NO:328-334, for example selected from SEQ ID NO:328-331 , for example selected from SEQ ID NO:328-329, for example SEQ ID NO:328.

7. HER3 binding polypeptide according to any preceding claim, which is capable of binding to HER3 such that the K_D value of the interaction with HER3 is at most 1 x 10^"8 M, such as at most 1 x 10^"9 M, such as at most

1 x 10^"10 M, such as at most 1 x 10^{"1 1} M.

8. HER3 binding polypeptide according to any preceding claim, which is capable of binding albumin.

9. HER3 binding polypeptide according to claim 8, wherein the HER3 binding polypeptide is capable of binding to albumin such that the K_D value of the interaction with albumin is at least 1 x 10^"8 M, such as at least 1 x 10^"7 M, such as at least 1 x 10^"6 M, such as at least 1 x 10^"5 M.

10. Fusion protein or conjugate comprising

a first moiety comprising of a HER3 binding polypeptide according to any preceding claim; and

a second moiety comprising of a polypeptide having a desired biological activity.

1 1 . Polynucleotide encoding a HER3 binding polypeptide or a fusion protein according to any one of claims 1 -10.

12. Expression vector comprising a polynucleotide according to claim

1 1 .

13. Host cell comprising an expression vector according to claim 12.

14. Method of producing a polypeptide or fusion protein according to any one of claims 1 -10, comprising

- culturing a host cell according to claim 13 under conditions permissive of expression of said polypeptide from its expression vector, and isolating said polypeptide.

15. Composition comprising a HER3 binding polypeptide, fusion protein or conjugate according to any one of claims 1 -10 and at least one pharmaceutically acceptable excipient or carrier.

16. HER3 binding polypeptide, fusion protein or conjugate according to any one of claims 1 -10 or composition according to claim 15 for use as a medicament.

17. HER3 binding polypeptide, fusion protein or conjugate according to any one of claims 1 -10 or composition according to claim 15 for use in diagnosis.

18. HER3 binding polypeptide, fusion protein or conjugate according to any one of claims 1 -10 or composition according to claim 15 for use in the treatment or diagnosis of a HER3 related condition, for example cancer, for example cancer selected from the group consisting of lung cancer, breast cancer, gastric cancer, stomach cancer, colon cancer, colorectal cancer, cancer of the small instestines, esophageal cancer, liver cancer, pancreas cancer, prostate cancer, kidney cancer, bladder cancer, ovarian cancer, uterine cancer, melanomas, cancers of the head and neck, pediatric gliomas, pediatric glioblastomas and astrocytomas.