WO2014076177A1 - Nouveau polypeptide - Google Patents

Nouveau polypeptide Download PDF

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Publication number
WO2014076177A1
WO2014076177A1 PCT/EP2013/073817 EP2013073817W WO2014076177A1 WO 2014076177 A1 WO2014076177 A1 WO 2014076177A1 EP 2013073817 W EP2013073817 W EP 2013073817W WO 2014076177 A1 WO2014076177 A1 WO 2014076177A1
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her3
sequence
binding polypeptide
seq
cancer
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PCT/EP2013/073817
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English (en)
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Sophia Hober
Mikael ÅSTRAND
John LÖFBLOM
Johan NILVEBRANT
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/76—Albumins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2318/00—Antibody mimetics or scaffolds
    • C07K2318/20—Antigen-binding scaffold molecules wherein the scaffold is not an immunoglobulin variable region or antibody mimetics

Definitions

  • 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.
  • the present disclosure relates to use of such a HER3 binding polypeptide as a diagnostic agent and/or a medicament.
  • 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.
  • EGFR ErbB1 or HER1
  • HER2 ErbB2
  • HER3 ErbB3
  • ErbB4 ErbB4
  • 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)).
  • HER3 has gained interest as an allosteric kinase activator of its family members.
  • HER2 and HER3 are 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)).
  • HER3 receptor 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 )).
  • 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)).
  • 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)).
  • the relatively large antibody molecule e.g. IgG
  • the extraordinarily long in vivo half-life of antibodies results in relatively high blood signals and thereby relatively poor tumor-to-blood contrasts.
  • 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);
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)).
  • ellers 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.
  • 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.
  • Streptococcus have also been identified, which contain domains similar to the albumin binding three-helix domains of protein G.
  • 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.
  • GA module protein G-related albumin binding module
  • a HER3 binding polypeptide comprising an amino acid sequence selected from: i) LAX3AKX6X7AX9X10 Xi i LDXi 4 Xi 5 GVSDX 2 o YKX 23 LIDKAKT
  • X3 is selected from D, Q, R, S and T;
  • Xe is selected from A, K, R and T;
  • X 7 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;
  • Xi 5 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;
  • X 43 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
  • the function of any polypeptide 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.
  • the invention encompasses modified variants of the HER3 binding polypeptide, which are such that the HER3 binding characteristics are retained.
  • the invention encompasses modified variants of the HER3 binding polypeptide which retain HER3 binding characteristics and albumin binding characteristics.
  • a HER3 binding polypeptide comprising an amino acid sequence with 93 % or greater identity to a polypeptide as defined in i).
  • 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).
  • 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).
  • an amino acid residue belonging to a certain functional grouping of amino acid residues e.g. hydrophobic, hydrophilic, polar etc
  • % identity 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
  • 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
  • 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.
  • X 3 in sequence i) is selected from R, S and T.
  • X 3 in sequence s selected from S and T is selected from S and T.
  • X3 in sequence i s selected from R and T is selected from R and T.
  • X 6 n sequence s selected from A, K and R In one embodiment X 6 n sequence s selected from K and R.
  • X 7 n sequence s selected from L and R.
  • X 7 n sequence In one embodiment Xg in sequence i) is L.
  • Xg in sequence i) is N.
  • Xio in sequence i) is selected from H and Y.
  • Xio in sequence i) is Y.
  • Xio in sequence i) is R.
  • Xi i in sequence i) is selected from F, I, L and V.
  • Xi i in sequence i) is selected from F, L and V.
  • Xi i in sequence i) is selected from I, L and V.
  • Xi i in sequence i) is selected from L and V.
  • Xi i in sequence i) is L.
  • Xi 4 in sequence i) is selected from A, D, G, K, L,
  • Xi 4 in sequence i) is selected from A, D, G, K, L,
  • Xi 4 in sequence i) is selected from D G, K, Q, R and S.
  • Xi 4 in sequence i) is selected from D G, K, R and
  • Xi 4 in sequence i) is selected from G K, Q, R and
  • Xi 4 in sequence i) is selected from D G, R and S.
  • Xi 4 in sequence i) is selected from G K, R, and S
  • Xi 4 in sequence i) is selected from G K and R.
  • Xi 4 in sequence i) is selected from G and R.
  • Xi 4 in sequence i) is selected from G and K.
  • Xi 4 in sequence i) is selected from S and R.
  • Xi 4 in sequence i) is G.
  • Xi 4 in sequence i) is R.
  • Xi 4 in sequence i) is K.
  • Xi 4 in sequence i) is S.
  • Xi 5 in sequence i) is selected from R T and V.
  • Xi 5 in sequence i) is selected from R and T.
  • Xi 5 in sequence i) is selected from R and V.
  • Xi 5 in sequence i) is selected from K and R.
  • Xi5 in sequence i) is R.
  • Xi 5 in sequence i) is T.
  • Xi 5 in sequence i) is V. In one embod ment Xl5 in sequence i ) is K.
  • ment X35 in sequence i ) is selected from H, Q and R.
  • ment X35 in sequence i ) is selected from H, M and R.
  • ment X35 in sequence i ) is selected from H, M and Q.
  • X43 in sequence i) is selected from A, G, H, I, P, R and V.
  • X43 in sequence is selected from A, G, R and V.
  • X43 in sequence is selected from G, R and V.
  • X43 in sequence is G.
  • X43 in sequence is V.
  • X43 in sequence is R.
  • X43 in sequence is selected from A, I, H and P.
  • X43 in sequence is selected from A, I and H.
  • X43 in sequence is selected from A and I.
  • X43 in sequence is A.
  • X43 in sequence is I.
  • X43 in sequence is H.
  • sequence i) is
  • ⁇ is selected from K and R;
  • Xg is selected from L and N;
  • X38 s selected from I, L and V;
  • 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.
  • 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.
  • sequence i) is selected from the group consisting of SEQ ID NO:1 -160.
  • 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.
  • said HER3 binding polypeptide is SEQ ID NO:1 .
  • sequence i) is selected from the group consisting of SEQ ID NO:161 -167.
  • sequence i) is selected from SEQ ID NO:161 -164, such as selected from SEQ ID NO:161 - 162.
  • sequence i) is SEQ ID NO:161 .
  • 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.
  • 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.
  • sequence i) is selected from the group consisting of SEQ ID NO:168-327.
  • 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.
  • sequence i) is SEQ IN NO:168.
  • sequence i) is selected from the group consisting of SEQ ID NO:328-334.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the HER3 binding polypeptide is capable of binding to HER3 such that the K D value of the interaction is at most
  • 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.
  • 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.
  • 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.
  • said albumin is human serum albumin.
  • any HER3 binding polypeptide disclosed herein may comprise further C terminal and/or N terminal amino acids.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the HER3 binding polypeptide of the invention may form homo- or heteromultimers, such as homo- or heterodimers.
  • one or more further polypeptide domain(s) may provide the HER3 binding polypeptide with another binding function.
  • 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
  • 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.
  • heteroogenic 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.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • 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.
  • 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.
  • 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.
  • labels may for example be used for detection of the polypeptide.
  • labeled polypeptide may for example be used for indirect labeling HER3 expressing tumors cells as well as metastatic cells.
  • 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.
  • an HER3 binding polypeptide comprising a second moiety, wherein said label is coupled to the second moiety only.
  • 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.
  • 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.
  • 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.
  • 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.
  • a chelator is the polyaminopolycarboxylate type of chelator.
  • Two classes of such polyaminopolycarboxylate chelators can be distinguished: macrocyclic and acyclic chelators.
  • the HER3 binding polypeptide, fusion protein or conjugate comprises a chelating environment provided by a
  • 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).
  • a chelating environment of the HER3 binding polypeptide, fusion protein or conjugate is provided by DOTA or a derivative thereof.
  • 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.
  • 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid (NOTA) and derivatives thereof may be used as chelators.
  • NOTA 1 ,4,7-triazacyclononane-1 ,4,7-triacetic acid
  • 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.
  • acyclic polyaminopolycarboxylate chelators are different derivatives of DTPA (diethylenetriamine-pentaacetic acid).
  • polypeptides having a chelating environment provided by
  • diethylenetriaminepentaacetic acid or derivatives thereof are also 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.
  • 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.
  • non-biological peptide synthesis using amino acids and/or amino acid derivatives having protected reactive side-chains, the non-biological peptide synthesis comprising
  • 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.
  • composition comprising a HER3 binding polypeptide, fusion protein or conjugate as described herein and at least one pharmaceutically acceptable excipient or carrier.
  • 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.
  • additional active agents include immunostimulatory agents, radionuclides, toxic agents, enzymes, factors recruiting effector cells (e.g. T or NK cells) and photosensitizers.
  • a HER3 binding polypeptide, fusion protein, conjugate or composition as described herein for use as a medicament there is provided a HER3 binding polypeptide, fusion protein .conjugate or
  • composition as described herein for use in diagnosis.
  • 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.
  • said HER3 related condition or cancer is further characterized by HER3 expression, over-expression and/or activation, e.g. hyperphosphorylation.
  • 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.
  • 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.
  • the HER3 binding polypeptide, fusion protein, conjugate or composition inhibits HER3 mediated signaling by binding to HER3 expressed on a cell surface.
  • the HER3 related condition is cancer.
  • 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
  • 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
  • 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)
  • Figure 2B is a schematic overview of the phage display selection of
  • FIG. 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.
  • 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- ⁇ ECD as described in Example 5. Obtained max responses were normalized against a
  • 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- ⁇ (heregulin).
  • Figure 7 shows a table representing the design of the affinity maturation library as described in Example 7.
  • 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.
  • FIG 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)
  • 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 H ER3-3 and non-randomized ABD.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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).
  • 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 .
  • 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 ⁇ bacterial stock with a cell density of 2.2 x 10 10 cfu/ml, corresponding to an approximate 100-fold excess of bacteria compared to the experimentally determined library size (Aim T el al, supra).
  • TTB tryptic soy broth
  • the culture volume was reduced to 100 ml.
  • the number of cells used for inoculation was always at least a 10 4 -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.
  • TSB+Y yeast extract
  • IPTG isopropyl ⁇ -D-l - thiogalactopyranoside
  • 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).
  • PEG polyethylene glycol
  • NaCI phosphate buffered saline
  • 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 ⁇ PBS supplemented with 0.1 % Tween 20 (PBST) and blocked for 20 min with 500 ⁇ PBS supplemented with 0.1 % (v/v) Tween 20 and 5 % (w/v) BSA (5 % PBSTB).
  • IgG-Fc was not included during pre-selection.
  • the entire phage-stock (10 11 phages) was used in cycle 1 and a 10 4 -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).
  • 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,
  • TST Tris-buffered saline
  • HSA human serum albumin
  • CV column volumes
  • 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
  • Binding kinetics for the seven selected ABD variants to albumin and HER3 were evaluated by surface plasmon resonance (SPR) using a
  • ProteOnTM XPR36 protein interaction array system BioRad, Hercules, CA, USA
  • Recombinant human HER3-Fc chimera catalog no. 348-RB, R&D Systems
  • His6-tagged human HER3 catalog no. 10201 -H08H, Sino Biological, Beijing, China
  • His 6 -tagged murine HER3 catalog no. 51003-M08H, mHER3; Sino Biological, Beijing, China
  • human serum albumin and murine serum albumin Sigma-Aldrich, St.
  • ABD3-3 SEQ ID NO:161
  • 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 ).
  • K D -values 10 and 12 nM, respectively
  • data from immobilized Fc-hHER3 and His6-hHER3 correlated well and therefore data from both versions of the receptor are presented together.
  • 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.
  • ABD3-2 166 1.0 ( ⁇ 0.2) x10 6 2.7 ( ⁇ 0.2) x10 "2 26 [4]
  • ABD3-3 161 8.3 ( ⁇ 0.2) x10 5 9.4 ( ⁇ 0.04) x10 "3 11 [2]
  • ABD3-3 SEQ ID NO:161
  • ABD3-27 SEQ ID NO:162
  • ABD3-20 SEQ ID NO:163
  • ABD3-18 SEQ ID NO:164
  • the negative control ABD SEQ ID NO:335
  • Human cat no. 348-RB, R&D Systems, Fc-fused,
  • murine cat no.
  • ABD3-27 lost the ability to bind hHER3 with HSA present at a 1 :1 molar ratio.
  • 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.
  • NRG- ⁇ ECD cat no.396-HB, R&D Systems
  • NRG- ⁇ EGF domain cat no. 377-HB, R&D Systems
  • 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- ⁇ .
  • a constant concentration (5 nM) of HER3 was incubated with a series of different concentrations of an ABD variant.
  • 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).
  • FBS fetal bovine serum
  • SKOV-3 cells (HER3
  • 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 10 5 - 7 x 10 5 cells were incubated with 500 nM ABD-Z at room
  • NRG- ⁇ EGF cat no. 396-HB, R&D Systems
  • 2 x 10 5 - 7 x 10 5 cells were incubated with 50 nM NRG- ⁇ 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).
  • 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.
  • ABD3-3 and ABD3-27 were further assessed on AU565-cells by pre-incubating the cells with NRG- ⁇ 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- ⁇ 1 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.
  • 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 10 7 and 4.4 x 10 6 variants on the nucleotide and protein level, respectively.
  • the library was produced as a 189 bp degenerate oligonucleotide (Integrated DNA
  • 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.
  • 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.
  • a dephosphorylation step (1 h, 37 °C; Antarctic Phosphatase, NEB
  • 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
  • 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).
  • 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.
  • the display vector and randomized library insert were successfully ligated.
  • 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.
  • Human Hise-tagged HER3 (cat no. 10201 -H08H, Sino Biological) was dissolved in PBS (150 mM NaCI, 8 mM Na 2 HPO 4 , 2 mM NaH 2 PO 4 ; 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,
  • FIG. 9 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
  • ODs78 1 ⁇ ⁇ 10 8 cells/ml, established by plating onto agar plates, ⁇ 10 8 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 ⁇ 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
  • Hulme and Trevethick Br. J. Pharmacol. 161 :1219-1237 (2010).
  • 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.
  • SAPE R-Phycoerythrin
  • Alexa Fluor 647 Invitrogen
  • 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.
  • >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
  • 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.
  • 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.
  • HER3 binding polypeptides were obtained 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.
  • variant 23_A01 SEQ ID NO:1
  • the said nine sequences exhibited a high level of % identity, differing only in three residue positions ( Figure 14).
  • 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 represent novel HER3 binding molecules with the favorable properties of small size and long serum half-life.
  • an off-rate selection step in which clones with slower dissociation kinetics are favored, was performed in two parallel tracks.
  • 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. 10 6 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 10 4 cells in a Gallios flow cytometer (Beckman Coulter, Brea, CA, USA) by measuring the median fluorescence intensity (MFI) of both SAPE and IgG- 647.
  • MFI median fluorescence intensity
  • 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).
  • 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.
  • variant 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.
  • Rosetta colonies were used to inoculate over-night cultures with TSB supplemented with 20 g/ml
  • 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.
  • HER3 Sesi Biological
  • serum albumin serum albumin
  • 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 ⁇ /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.
  • 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.
  • 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.
  • HER3 binding polypeptide comprising an amino acid sequence selected from: i) LAX3AKX6X7AX9X10 XiiLDXi 4 Xi 5 GVSDX 2 o YKX 23 LIDKAKT
  • 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;
  • 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;
  • 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;
  • 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).
  • HER3 binding polypeptide according to item 1 wherein X 3 in sequence i) is selected from R, S and T. 3. HER3 binding polypeptide according to item 2, wherein X 3 in sequence i) is selected from S and T.
  • HER3 binding polypeptide according to item 2 wherein X 3 in sequence i) is selected from R and T.
  • HER3 binding polypeptide according to item 3 wherein X 3 in sequence i) is S.
  • HER3 binding polypeptide according to any preceding item, wherein Xe in sequence i) is selected from A, K and R.
  • HER3 binding polypeptide according to item 8 wherein X 6 in sequence i) is selected from K and R.
  • HER3 binding polypeptide according to item 9 wherein X 6 in sequence i) is R. 1 1 .
  • HER3 binding polypeptide according to any preceding item, wherein X 7 in sequence i) is selected from L and R.
  • HER3 binding polypeptide according to any preceding item, wherein X 9 in sequence i) is L.
  • HER3 binding polypeptide according to item 16 wherein Xi 0 in sequence i) is Y.
  • HER3 binding polypeptide according to any one of items 1 -15, wherein Xi 0 in sequence i) is R.
  • HER3 binding polypeptide according to any preceding item, wherein X in sequence i) is selected from F, I, L and V.
  • HER3 binding polypeptide according to item 19, wherein X in sequence i) is selected from F, L and V.
  • 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.
  • 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.
  • 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.
  • HER3 binding polypeptide according to item 31 wherein Xi in sequence i) is selected from G and R.
  • HER3 binding polypeptide according to any preceding item wherein Xi 5 in sequence i) is selected from R, T and V. 40. HER3 binding polypeptide according to item 39, wherein Xi 5 in sequence i) is selected from R and T.
  • HER3 binding polypeptide according to item 41 wherein Xi 5 in sequence i) is V.
  • HER3 binding polypeptide according to any preceding item, wherein X 2 3 in sequence i) is D.
  • HER3 binding polypeptide according to any one of items 1 -48, wherein X 2 3 in sequence i) is R.
  • HER3 binding polypeptide according to any preceding item wherein X 35 in sequence i) is selected from H, Q and R. 52.
  • HER3 binding polypeptide according to any one of items 1 -50, wherein X 35 in sequence i) is selected from H, M and R.
  • HER3 binding polypeptide according to any one of items 51 and 55, wherein X 35 in sequence i) is H.
  • HER3 binding polypeptide according to any one of items 52-53, wherein X 35 in sequence i) is R.
  • HER3 binding polypeptide according to item 61 wherein X 38 in sequence i) is selected from I and V.
  • HER3 binding polypeptide according to item 61 wherein X 38 in sequence i) is selected from I and L. 64. HER3 binding polypeptide according to item 61 , wherein X 38 in sequence i) is selected from L and V.
  • HER3 binding polypeptide according to any one of items 62-63, wherein X 38 in sequence i) is I.
  • HER3 binding polypeptide according to any one of items 62, 64 and 68, wherein X 38 in sequence i) is V.
  • HER3 binding polypeptide according to any preceding item, wherein X 39 is selected from F, I, L and R.
  • 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.
  • HER3 binding polypeptide according to item 81 wherein X43 in sequence i) is selected from A, G, R and V.
  • 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.
  • 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;
  • X43 is selected from G, I, R and V.
  • 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.
  • sequence i) is selected from SEQ ID NO:1 -167 and 338-387, such as selected from SEQ ID NO:1 -167.
  • sequence i) is selected from SEQ ID NO:1 -160.
  • HER3 binding polypeptide according to item 98, wherein sequence i) is selected from SEQ ID NO:161 -164.
  • 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 .
  • 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.
  • HER3 binding polypeptide according to item 105 wherein sequence i) is selected from SEQ ID NO:168-176.
  • sequence i) is selected from SEQ ID NO:328-334.
  • HER3 binding polypeptide according to item 109, wherein sequence i) is SEQ ID NO:328.
  • HER3 binding polypeptide according to item 105, wherein sequence i) is selected from SEQ ID NO:168-176, 221 , 250 and 253.
  • 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.
  • HER3 binding polypeptide according to any one of items 1 14-1 15, wherein said albumin is human serum albumin.
  • HER3 binding polypeptide according to any preceding item as a multimer, such as a dimer. 1 18. Fusion protein or conjugate comprising
  • 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.
  • 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 analgou
  • HER3 binding polypeptide, fusion protein or conjugate according to any preceding item further comprising a label.
  • said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.
  • 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.
  • 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.
  • Host cell comprising an expression vector according to item 135.
  • 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.
  • composition according to item 138 further comprising at least one additional active agent.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne une classe de polypeptides techniques présentant une affinité de liaison pour le récepteur du facteur de croissance épidermique humain 3 (HER3), et concerne un polypeptide de liaison à HER3 comprenant la séquence LAX3AKX6X7AX9X10 X11LDX14X15GVSDX20 YKX23LIDKAKT VEGVX35ALX38X39X40 ILX43ALP. La présente invention concerne également ledit polypeptide de liaison à HER3 présentant également une affinité pour l'albumine. Par ailleurs, la présente invention concerne l'utilisation d'un tel polypeptide de liaison à HER3 en tant qu'agent de diagnostic et/ou médicament.
PCT/EP2013/073817 2012-11-14 2013-11-14 Nouveau polypeptide WO2014076177A1 (fr)

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EP3904378A1 (fr) * 2020-04-29 2021-11-03 Hober Biotech AB Protéine bispécifique
CN116785409A (zh) * 2023-08-23 2023-09-22 青岛汇丰动物保健品有限公司 一种多肽在制备提高家禽肠道健康的兽药中的应用

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019175176A1 (fr) 2018-03-13 2019-09-19 Affibody Ab Polypeptides basés sur un nouvel échafaudage
EP3904378A1 (fr) * 2020-04-29 2021-11-03 Hober Biotech AB Protéine bispécifique
WO2021219818A1 (fr) * 2020-04-29 2021-11-04 Hober Biotech Ab Protéine bispécifique
CN116785409A (zh) * 2023-08-23 2023-09-22 青岛汇丰动物保健品有限公司 一种多肽在制备提高家禽肠道健康的兽药中的应用
CN116785409B (zh) * 2023-08-23 2023-11-07 青岛汇丰动物保健品有限公司 一种多肽在制备提高家禽肠道健康的兽药中的应用

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