Specific binding proteins for human erb-b3 receptor tyrosine kinase 3 (HER3)

Abstract

The present invention relates to new proteins that are specific for human epidermal growth factor kinase receptor 3 (ERB-B3 receptor tyrosine kinase 3, Her3). The Her3 specific proteins of the invention bind with high affinity to human Her3. The invention further refers to Her3 specific proteins that further comprise a diagnostically or therapeutically active component. Further aspects of the invention cover the use of these Her3 specific binding proteins in medicine, for example, in diagnosis and therapy of Her3 related cancer.

Description

[0001] SPECIFIC BINDING PROTEINS FOR HUMAN ERB-B3 RECEPTOR TYROSINE KINASE 3 (HER3)

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to new proteins that are specific for human epidermal growth factor kinase receptor 3 (ERB-B3 receptor tyrosine kinase 3, Her3). The Her3 specific proteins of the invention bind with high affinity to human Her3. The invention further refers to Her3 specific proteins that further comprise a diagnostically or therapeutically active component. Further aspects of the invention cover the use of these Her3 specific binding proteins in medicine, for example, in diagnosis and therapy of Her3 related cancer.

[0004] BACKGROUND OF THE INVENTION

[0005] Human epidermal growth factor receptor 3 (Her3) is a unique member of the human epidermal growth factor receptor family. Her3 is a catalytically defective receptor tyrosine kinase and has only weak oncogenic power by itself. Only after interaction with a kinase-proficient receptor of the receptor tyrosine kinase family for example forming a heterodimer Her2-Her3, signaling cascades are activated by interacting with Her2. The activation of signaling kinases promotes oncogenesis, metastatic dissemination, and drug resistance. Her3 in complex with Her2 is implicated in growth, proliferation, chemotherapeutic resistance and promotion of invasion and metastasis.

[0006] Approaches to prevent dimerization of Her3 with Her2 were addressed by therapeutically targeting Her3 with monoclonal antibodies or bispecific antibodies. However, this unfortunately led to unsatisfactory results across several tumor types.

[0007] Diagnosis and treatment of Her3 related cancer is often not adequately addressed by existing options, and as a consequence, many patients do not adequately benefit from current strategies. Up to date, no Her3-targeted therapy has been approved for cancer treatment. Needless to say, that there is a strong need for novel strategies for diagnosis and treatment of tumors with Her3 overexpression, as well as other Her3 related diseases and disorders.

[0008] One objective of the present invention is the provision of proteins for specific targeting of Her3 for allowing targeted diagnostic and treatment options, including detection of Her3 positive tumors (e.g. by radio-diagnostic methods). Targeting this tumor-associated protein may offer benefit to patients with unmet need for novel diagnostic and therapeutic routes. Targeting Her3 suggests a potentially non-toxic diagnostic and treatment approach, due to low and restricted distribution of Her3 in normal tissues. Thus, binding proteins with specificity for Her3 may enable effective medical options for cancer treatment, and finally improve quality of life for patients. The invention provides novel Her3 binding molecules for new and improved strategies in the diagnosis and treatment of Her3 positive tumors.

[0009] The above-described objectives and advantages are achieved by the subject-matter of the appended claims. The present invention meets the needs presented above by providing novel high affinity specific Her3 binding proteins. The above overview does not necessarily describe all problems solved by the present invention.

[0010] SUMMARY OF THE INVENTION

[0011] The present disclosure provides the following items 1 to 16, without being specifically limited thereto:

[0012] 1. A binding protein for human Her3 comprising

[0013] (a) an amino acid sequence with at least 80 % identity to SEQ ID NO: 1, wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and

[0014] (b) wherein the Her3 binding protein has a binding affinity for Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR). Preferably, the Her3 binding protein has binding affinity of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, for the extracellular domain (ECD-Fc) of human Her3, as determined by Surface Plasmon Resonance (SPR).

[0015] 2. The binding protein for human Her3 according to claim 1 wherein the amino acid in position 74 of SEQ ID NO: 1 is Alanine (A).

[0016] 3. The binding protein for human Her3 according to item 1 or item 2 wherein the amino acid in position 76 of SEQ ID NO: 1 is Leucine (L).

[0017] 4. The binding protein for human Her3 according to items 1-3, comprising an amino acid sequence selected from any of SEQ ID NOs: 1-31.

[0018] 5. The binding protein for human Her3 according to items 1-4, wherein the protein has no detectable binding affinity to Her2.

[0019] 6. A multimer comprising the binding protein for human Her3 according to items 1-5 wherein the multimer is a dimer, a trimer, a tetramer, a pentamer, or a hexamer, preferably a dimer.

[0020] 7. The binding protein according to items 1-6, wherein the binding protein comprises additionally at least one molecule modulating pharmacokinetics selected optionally from an albumin-binding protein, an albumin binding small molecule, a serum albumin, an immunoglobulin binding protein, an immunoglobulin or immunoglobulin fragment, a polysaccharide, an unstructured amino acid sequence comprising amino acids alanine, glycine, serine, proline; a polyethylene glycol, a sialic acid, a transferrin, a fatty acid, and a transferrin receptor binding protein, or any combination thereof.

[0021] 8. The binding protein according to items 1-7, wherein the binding protein comprises additionally at least one diagnostically active moiety, optionally selected from a radionuclide, a chelator with a radionuclide, fluorescent protein, photosensitizer, dye, or enzyme, or any combination of the above,

[0022] 9. The binding protein according to items 1-7, wherein the binding protein comprises additionally at least one therapeutically active moiety, optionally selected from a monoclonal antibody or a fragment thereof, a binding protein, a receptor or receptor domain, a radionuclide, a chelator with a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or derivatives thereof, or any combination of the above.

[0023] 10. The binding protein according to items 1-9, for use in diagnostics or treatment of cancer.

[0024] 11. A nucleic acid molecule encoding the binding protein according to items 1-9.

[0025] 12. A vector comprising the nucleic acid molecule of item 11.

[0026] 13. A host cell or a non-human host comprising the binding protein according to items 1- 9, a nucleic acid as defined in item 11 , and / or a vector of item 12.

[0027] 14. A composition comprising the binding protein according to items 1-9 for use in medicine, preferably for use in the diagnosis or treatment of Her3 positive tumors or cancer cells.

[0028] 15. A method of producing the binding protein according to items 1-9 comprising a) culturing a host cell of item 13 under suitable conditions in order to obtain said binding protein and b) isolating said binding protein and optionally isolating said binding protein from the host cell of item 13 and / or the medium in which the host cell of item 13 is growing.

[0029] 16. A method of detecting Her3 in a sample, the method comprising (a) providing a sample, (b) providing the binding protein according to items 1-9, (c) contacting the binding protein with the sample under conditions that permit binding of the binding protein according to items 1-9; and (d) determine the binding to Her3 in the sample.

[0030] This summary does not necessarily describe all features of the present invention. Other embodiments come apparent from a review of the ensuing detailed description.

[0031] BRIEF DESCRIPTION OF THE FIGURES

[0032] The Figures show: FIGURE 1 : shows the binding affinity of Her3 binding protein after long-term incubation in human serum. After 24 h incubation in serum, the binding affinity (KD value) to Her3 expressed on cells showed only minor variation. The binding affinity of SEQ ID NO: 1 to Her3 after incubation in mouse serum is shown in FIG. 1A. The binding affinity of SEQ ID NO: 1 to Her3 after incubation in human serum is shown in FIG. 1B. The results confirm the stability of Her3 binding proteins. Even after long term incubation in human serum, the Her3 binding proteins bind with high affinity to Her3 expressed on cells.

[0033] DETAILED DESCRIPTION OF THE INVENTION

[0034] The present inventors have developed a solution to meet the strong ongoing need in the art for expanding medical options for the diagnosis and treatment of diseases by providing novel Her3 binding proteins. The Her3 specific proteins as defined herein are functionally characterized by high specific affinity for (human) Her3 of less than 30 nM. Further, they show stability in serum. The novel proteins as described herein may broaden so far unmet medical strategies for the diagnosis and therapy of Her3 related cancer. In particular, the Her3 binding protein as described herein may be used for imaging purposes, for example, for the presence of tumor cells expressing Her3, and for radiotherapy treatment of tumors expressing Her3.

[0035] Before the present invention is described in more detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention which is reflected by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. This includes a skilled person working in the field of developing new target-specific binding molecules for use in technical applications and in therapy and diagnostics.

[0036] Preferably, the terms used herein are defined as described in “A multilingual glossary of biotechnological terms: (IUPAC Recommendations)”, Leuenberger, H.G.W, Nagel, B. and Kolbl, H. eds. (1995), Helvetica Chimica Acta, CH-4010 Basel, Switzerland).

[0037] Throughout this specification and the claims, which follow, unless the context requires otherwise, the word “comprise”, and variants such as “comprises” and “comprising”, was understood to imply the inclusion of a stated integer or step, or group of integers or steps, but not the exclusion of any other integer or step or group of integers or steps. The term “comprise(s)” or “comprising” may encompass a limitation to “consists of” or “consisting of”, should such a limitation be necessary for any reason and to any extent.

[0038] Several documents (for example: patents, patent applications, scientific publications, manufacturer’s specifications, instructions, GenBank Accession Number sequence submissions etc.) may be cited throughout the present specification. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. Some of the documents cited herein may be characterized as being “incorporated by reference". In the event of a conflict between the definitions or teachings of such incorporated references and definitions or teachings recited in the present specification, the text of the present specification takes precedence.

[0039] All sequences referred to herein are disclosed in the attached sequence listing that, with its whole content and disclosure, forms part of the disclosure content of the present specification.

[0040] GENERAL DEFINITIONS OF IMPORTANT TERMS USED IN THE APPLICATION

[0041] The term “Her3“ as used herein refers to Uniprot accession number P21860 (human Her3, alternative name: erb-b2 receptor tyrosine kinase 3). The term „Her3” comprises all polypeptides which show a sequence identity of at least 70 %, 75 %, 80 %, 85 %, 90 %, 95 %, 96 % or 97 % or more, or 100 % to human Her3 of Uniprot accession number P21860.

[0042] The term "Her3 binding protein" or “binding protein for Her3” refers to a protein of the present invention with high affinity binding to Her3. The term "Her3 binding protein" or “binding protein for Her3” also refers to a protein of the present invention with high affinity binding to the extracellular domain of human Her3 (such as ECD-Fc fusion hHer3). As described elsewhere herein, the proteins of the present disclosure exhibit a specific binding affinity for human Her3, in particular to the extracellular domain of human Her3, which is a binding affinity in the nanomolar range.

[0043] The terms “protein” and “polypeptide” refer to any chain of two or more amino acids linked by peptide bonds, and does not refer to a specific length of the product. Thus, “peptides”, “protein”, “amino acid chain”, or any other term used to refer to a chain of two or more amino acids, are included within the definition of “polypeptide”, and the term “polypeptide” may be used instead of, or interchangeably with, any of these terms. The term “polypeptide” is also intended to refer to the products of post-translational modifications of the polypeptide, which are well known in the art.

[0044] The term "modification" or "amino acid modification" refers to a substitution, a deletion, or an insertion of a reference amino acid at a particular position in a parent polypeptide sequence by another amino acid. Given the known genetic code, and recombinant and synthetic DNA techniques, the skilled scientist can readily construct DNAs encoding the amino acid variants. The term “amino acid substitution” is understood as an exchange of an amino acid by another amino acid.

[0045] The terms “binding affinity” and “binding activity” may be used herein interchangeably, and they refer to the ability of a polypeptide to bind to another protein, peptide, or fragment or domain thereof. Binding affinity is typically measured and reported by the equilibrium dissociation constant (KD), which is used to evaluate and rank order strengths of biomolecular interactions.

[0046] The term “fusion protein” relates to a protein comprising at least a first protein joined genetically to at least a second protein. A fusion protein is created through joining of two or more genes that originally coded for separate proteins. Fusion proteins may further comprise additional domains that are not involved in binding of the target, such as but not limited to, for example, multimerization moieties, polypeptide tags, polypeptide linkers or moieties binding to a target different from Her3.

[0047] The term “amino acid sequence identity” refers to a quantitative comparison of the identity (or differences) of the amino acid sequences of two or more proteins. “Percent (%) amino acid sequence identity” with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. To determine the sequence identity, the sequence of a query protein is aligned to the sequence of a reference protein or polypeptide. Methods for sequence alignment are well known in the art. For example, for determining the extent of an amino acid sequence identity of an arbitrary polypeptide relative to another amino acid sequence, the SIM Local similarity program as known in the art is preferably employed. For multiple alignment analysis, Clustal Omega is preferably used, as known to someone skilled in the art.

[0048] DETAILED DESCRIPTION OF THE EMBODIMENTS OF THIS INVENTION

[0049] Structural characterization of Erb-b2 receptor tyrosine kinase 3 (Her3) binding proteins.

[0050] The binding protein for human Her3 as described herein comprises an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 wherein the binding protein has a threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1.

[0051] In various embodiments, the binding protein for human Her3 is selected from amino acid sequences with at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92 %, 93 %, 94 %, 95%, 96 %, 97 %, 98%, or 99% identity to the amino acid sequence of SEQ ID NO: 1 wherein the binding protein has a threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1.

[0052] The binding protein for human Her3 as described herein comprises an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 wherein the binding protein has a threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , and has a binding affinity for human Her3 of at least (or less than) 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0053] Specific examples for the binding protein for human Her3 as described herein that comprise an amino acid sequence with at least 80 % identity, at least 85 % identity, at least 90 % identity, at least 95 % identity to the amino acid sequence of SEQ ID NO: 1 are provided in SEQ ID NOs: 1-31. In some embodiments, binding proteins for human Her3 comprise an amino acid sequence selected from any of SEQ ID NOs: 1-31 . In some embodiments, binding proteins for the extracellular domain of human Her3 comprise an amino acid sequence selected from any of SEQ ID NOs: 1-31.

[0054] As a general concept, the present invention provides a binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0055] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 and alanine (A) at the position corresponding to position 74 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0056] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0057] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , alanine (A) at the position corresponding to position 74 of SEQ ID NO: 1, and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0058] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , and lysine (K), valine (V), glutamine (Q) or arginine (R) at the position corresponding to position 6 of SEQ ID NO: 1 , alanine (A) at the position corresponding to position 74 of SEQ ID NO: 1 , and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0059] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , and lysine (K), valine (V), glutamine (Q) or arginine (R) at the position corresponding to position 6 of SEQ ID NO: 1 , lysine (K), tyrosine (Y), arginine (R), or isoleucine (I) at the position corresponding to position 54 of SEQ ID NO: 1 , alanine (A), or glycine (G) at the position corresponding to position 74 of SEQ ID NO: 1 , and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for hHer3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0060] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , lysine (K), valine (V), glutamine (Q), or arginine (R) at the position corresponding to position 6 of SEQ ID NO: 1 , tryptophane (W) or tyrosine (Y) at the position corresponding to position 51 of SEQ ID NO: 1 , alanine (A) at the position corresponding to position 74 of SEQ ID NO: 1 , and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0061] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , threonine (T) or alanine (A) at the position corresponding to position 7 of SEQ ID NO: 1 , alanine (A) at the position corresponding to position 74 of SEQ ID NO: 1 , and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0062] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , lysine (K), valine (V), glutamine (Q) or arginine (R) at the position corresponding to position 6 of SEQ ID NO: 1 , aspartic acid (D) or tyrosine (Y) at the position corresponding to position 45 of SEQ ID NO: 1 , alanine (A) at the position corresponding to position 74 of SEQ ID NO: 1 , and leucine (L) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0063] In various other embodiments, the binding protein for human Her3 comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 has threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1 , alanine (A) or glycine (G) at the position corresponding to position 74 of SEQ ID NO: 1 , and optionally at least one or two or three or more amino acids of the following:

[0064] • lysine (K), valine (V), glutamine (Q), or arginine (R) at the position corresponding to position 6 of SEQ I D NO: 1 ,

[0065] • threonine (T) or alanine (A) at the position corresponding to position 7 of SEQ ID NO: 1 ,

[0066] • glutamic acid (E) or glycine (G) at the position corresponding to position 30 of SEQ ID NO: 1 ,

[0067] • arginine (N), lysine (K) or tyrosine (Y) at the position corresponding to position 31 of SEQ ID NO: 1,

[0068] • glutamine (Q) or arginine (R) at the position corresponding to position 37 of SEQ ID NO: 1 ,

[0069] • proline (P) or serine (S) at the position corresponding to position 44 of SEQ ID NO: 1 ,

[0070] • aspartic acid (D) or tyrosine (Y) at the position corresponding to position 45 of SEQ ID NO: 1 ,

[0071] • arginine (R) or leucine (L) at the position corresponding to position 48 of SEQ ID NO: 1 ,

[0072] • isoleucine (I) or tryptophan (W) or valine (V) at the position corresponding to position 50 of SEQ ID NO: 1 ,

[0073] • tryptophan (W) or tyrosine (Y) at the position corresponding to position 51 of SEQ ID NO: 1 ,

[0074] • lysine (K) or tyrosine (Y) or arginine (R) or isoleucine (I) at the position corresponding to position 54 of SEQ ID NO: 1 ,

[0075] • aspartic acid (D) or glycine (G) at the position corresponding to position 64 of SEQ ID NO: 1 , and / or

[0076] • leucine (L) or glycine (G) at the position corresponding to position 76 of SEQ ID NO: 1 , and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, more preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR). In the herein-described embodiments concerning the binding protein for human Her3 of SEQ ID NO: 1 , including in particular the herein-described embodiments concerning threonine or serine at the position corresponding to position 10 of SEQ ID NO: 1 , the binding protein for human Her3 may comprise an amino acid sequence with at least 85 % identity to the amino acid sequence of SEQ ID NO: 1. Preferably, the binding protein for human Her3 may comprise an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 1. More preferably, the binding protein for human Her3 may comprise an amino acid sequence with at least 95 % identity to the amino acid sequence of SEQ ID NO: 1. Still more preferably, the binding protein for human Her3 may comprise an amino acid sequence with at least 96 % identity to the amino acid sequence of SEQ ID NO: 1. Even more preferably, the binding protein for human Her3 may comprise an amino acid sequence with at least 97 % or at least 98 % identity to the amino acid sequence of SEQ ID NO: 1 .

[0077] As further described herein, a binding protein for human Her3 comprises an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 and may in various embodiments describe a variant amino acid sequence (solely) based on amino acid substitutions as compared to the reference sequence(s) disclosed herein, in particular the amino acid sequence of SEQ ID NO: 1. Such binding protein for human Her3 has a length of about 82 amino acids. In other embodiments, a binding protein of Her3 that comprises an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 may in various embodiments describe a variant amino acid sequence (solely) based on amino acid substitutions as compared to the reference sequence disclosed herein, in particular the sequence of SEQ ID NO: 1.

[0078] The herein-described embodiments concerning SEQ ID NO: 1 , including in particular the herein-described embodiments concerning threonine or serine at the position corresponding to position 10 of SEQ ID NO: 1, also apply to multimeric forms disclosed herein comprising a protein comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), as described herein, and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, more preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

[0079] The binding protein for human Her3 disclosed herein, including multimeric forms thereof, bind to or are considered to bind specifically to Her3. In preferred embodiments, the binding protein for human Her3 does not bind to human Her2.

[0080] Functional characterization. The Her3 binding proteins as described herein, including multimeric forms thereof disclosed herein, have a binding affinity (KD) of less than (at least) 30 nM for human Her3, preferably to the extracellular domain of human Her3, preferably to ECD-Fc of Her3. In various embodiments, the protein binds human Her3 with measurable binding affinity of any of less than 10 nM, and / or even less than 6 nM (as determined by SPR). As described elsewhere herein, the lower the KD value, the greater the binding affinity of the biomolecule for its binding partner. The higher the KD value, the more weakly the binding partners bind to each other (see Examples). Accordingly, as described herein, the terms Jess than 30 nM” and “at least 30 nM” may be used interchangeably herein since they both refer to the high binding affinity of the proteins of the invention for Her3. More specifically, in the context of high binding affinity, the terms “less than 30 nM” and “at least 30 nM” each mean a range of numerical values indicating KD values < 30 nM (= including 30 nM) because the lower the KD value, the greater the binding affinity. The same considerations with regard to the interchangeable use of the terms Jess than” and “at least” apply with regard to all preferred binding affinities for Her3 disclosed throughout the specification, e.g., Jess than 30 nM” and “at least 30 nM” etc.. Here again, the terms “less than 30 nM” and “at least 30 nM” each mean a range of numerical values indicating KD values < 30 nM (= including 30 nM) because the lower the KD value, the greater the binding affinity.

[0081] In preferred embodiments, the binding protein for human Her3, including multimeric (e.g. dimeric) forms thereof disclosed herein, binds human Her3 (e.g. to the extracellular domain of human Her3) with measurable binding affinity of less than 10 nM. In various embodiments of the present invention, the binding affinity for Her3 of less than 30 nM as described above and throughout the specification means a binding affinity for human Her3 (hHer3) of less than (at least) 30 nM. Preferred binding affinities (less than 10 nM etc.) are described above and elsewhere herein and apply to embodiments in relation to binding of the proteins and multimers of the present invention to hHer3.

[0082] In some embodiments, the binding protein for human Her3, including multimeric (e.g. dimeric) forms thereof disclosed herein, do not bind to human Her2 with measurable binding affinity. The appropriate methods are known to those skilled in the art or described in the literature. The methods for determining the binding affinities are known per se and can be selected for instance from the following methods known in the art: enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), kinetic exclusion analysis (KinExA assay), Biolayer interferometry (BLI), flow cytometry, fluorescence spectroscopy techniques, isothermal titration calorimetry (ITC), analytical ultracentrifugation, radioimmunoassay (RIA or IRMA), and enhanced chemiluminescence (ECL). Some of the methods are described in the Examples below. Typically, the dissociation constant KD is determined at 20°C, 25°C, or 30°C. If not specifically indicated otherwise, the KD values recited herein are determined at 25°C by SPR. The lower the KD value, the greater the binding affinity of the biomolecule for its binding partner. The higher the KD value, the moreweakly the binding partners bind to each other (see Examples). As disclosed herein, in the above-described embodiments, the binding protein may comprise an amino acid sequence with at least 80 % identity or at least 85 % identity or at least 90 % identity or at least 95 % identity to the amino acid sequence of SEQ ID NO: 1 ,and exhibits a binding affinity for Her3 of less than 30 nM. As further disclosed herein, in the above-described embodiments, the binding protein may comprise an amino acid sequence with at least 80 % identity or at least 85 % identity or at least 90 % identity or at least 95 % identity to the amino acid sequence of SEQ ID NO: 1 ,and exhibits a binding affinity for Her3 of less than 10 nM. The binding protein may comprise an amino acid sequence with at least 80 % identity or at least 85 % identity or at least 90 % identity or at least 95 % identity to the amino acid sequence of SEQ ID NO: 1 and exhibits a binding affinity for Her3 of less than 6 nM.

[0083] As still further disclosed herein, in the embodiments concerning SEQ ID NO: 1 described above, the binding protein, including multimeric (dimeric) forms thereof disclosed herein, preferably exhibits a binding affinity for Her3 of less than 30 nM. More preferably, the protein including multimeric forms thereof disclosed herein exhibits a binding affinity for Her3 of less than 10 nM. Even more preferably, the protein including multimeric forms thereof disclosed herein exhibits a binding affinity for Her3 of less than 6 nM.

[0084] The herein-described embodiments concerning functional characterization of the Her3 binding protein of the invention also apply to multimeric forms disclosed herein comprising a protein comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 as described herein.

[0085] Binding of the protein as described herein to cellular Her3 can be determined by standard methods, including Immunofluorescence microscopy and flow cytometric analysis. In some embodiments, specific Her3 binding of the proteins of the present invention is determined by cellular Her3 binding analysis. In some embodiments, the Her3 binding proteins of the invention show binding to Her3 on Her3 expressing cells (see Examples). In some embodiments, the Her3 binding proteins of the invention exhibit a binding affinity for Her3 of less than 1 nM on Her3 expressing cells (see Examples). More specifically, in various embodiments the Her3 binding proteins of the invention preferably exhibit a binding affinity for Her3 expressed on cells of less than 1 nM (see Examples).

[0086] In some embodiments, the Her3 binding proteins as described herein is particularly stable under different conditions. In preferred embodiments, the Her3 binding proteins is stable in the presence of (human or mouse) serum for at least 24 h at 37 °C. In some embodiments, the Her3 binding proteins as described herein is stable in the presence of human serum for at least 24 h at 37 °C, as described in Example 6 in more detail. In some embodiments, the protein as described herein, is stable in the presence of mouse serum for at least 24 h at 37 °C, as described in Example 6 in more detail. For example, the stability of a Her3 binding protein can be determined by measuring the binding affinity (KD) after incubation in serum for a long period of time at temperatures as high as 37 °C using standard methods as described herein above and in the Examples. In the embodiments described above, the Her3 binding proteins preferably exhibit a binding affinity for Her3 of less than about 20 nM, preferably less than about 10 nM, more preferably less than about 5 nM, even more preferably less than about 1 nM on Her3 expressing cells, even after incubation in (human) serum for 24 h at 37 °C. More specifically, in various embodiments the protein preferably exhibits a binding affinity for Her3 of less than about 20 nM, preferably less than about 10 nM, more preferably less than about 5 nM, even more preferably less than about 1 nM on Her3 expressing cells as determined by flow cytometry analysis, even after incubation in (human) serum for 24 h at 37 °C.

[0087] Also, the above-described further embodiments concerning functional characterization of the Her3 binding protein of the invention apply to multimeric forms disclosed herein comprising a protein comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 described herein throughout.

[0088] Multimers. The present invention provides multimers comprising a protein of the present invention as described above and throughout the present specification, / .e., as a general concept, the present invention provides a multimer comprising a protein comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and wherein the binding protein has a binding affinity for Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by SPR.

[0089] More specifically, the present invention provides a multimer comprising a protein as described elsewhere herein in relation to embodiments concerning threonine (T) or serine (S) at the position corresponding to position 10 of SEQ ID NO: 1.

[0090] In the present invention, the multimer may be any of a dimer, a trimer, a tetramer, a pentamer, or a hexamer. In the present invention, the multimer preferably is a dimer or a trimer, more preferably a dimer. Accordingly, a multimer of the present invention comprises at least two monomers (= dimer). Accordingly, a protein of the present invention as described above throughout the present specification may be considered as a protein monomer of a multimer provided by the present invention. Such protein monomers have a length of or comprise about 82 amino acids.

[0091] In some embodiments, a multimer may comprise one, two, three, four, or more protein(s) as described herein. In one embodiment, the protein comprises 2, 3, 4, or more proteins linked to each other, i.e. the protein can be a dimer, trimer, or tetramer, etc. as described above, wherein at least one protein of the multimer is comprising an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and wherein the binding protein has a binding affinity for Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by SPR. In one preferred embodiment, the Her3 binding protein comprises 2 proteins linked to each other wherein one protein of the multimer is comprising an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and wherein the binding protein has a binding affinity for Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by SPR.

[0092] In various preferred embodiments of the present invention, the multimer comprises a protein of the present invention as described above comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1. In preferred embodiments, the multimer of the present invention comprises at least two protein domains. In one preferred embodiment, the Her3 binding protein comprises 2 proteins linked to each other wherein the protein comprising an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and wherein the binding protein has a binding affinity for Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by SPR.

[0093] In various embodiments, the multimer comprises a protein of the present invention as described above and throughout the present specification, and a binding protein comprising an amino acid sequence with at least 80 % identity to the amino acid sequence of 229627 (SEQ ID NO: 6; dimer of SEQ ID NO: 1), 229629 (SEQ ID NO: 7; dimer of SEQ ID NO: 2), or229625 (SEQ ID NO: 5, dimer of SEQ ID NO: 4). In various preferred embodiments, the multimer comprises a binding protein having an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1. More preferably, the multimer comprises a binding protein for Her3 having an amino acid sequence with at least 85 % identity to the amino acid sequence of SEQ ID NO: 1. Still more preferably, the multimer comprises a binding protein having an amino acid sequence with at least 90 % identity to the amino acid sequence of SEQ ID NO: 1. Even more preferably, the multimer comprises a binding protein having an amino acid sequence with at least 95 % identity to the amino acid sequence of SEQ ID NO: 1. In particularly preferred embodiments, the multimer comprises a binding protein having an amino acid sequence with at least 98 % or even 100 % identity to the amino acid sequence of SEQ ID NO: 1.

[0094] Preferably, the multimer is a dimer comprising two identical or non-identical binding proteins having an amino acid sequence with at least 80 % identity to the amino acid sequence of SEQ ID NO: 1.

[0095] Preferred embodiments relate to a dimeric Her3 binding protein comprising and an amino acid sequence with at least 80 % identity to amino acid sequence of SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and wherein the binding protein exhibits a binding affinity for human Her3 of less than 30 nM as determined by Surface Plasmon Resonance.

[0096] In some embodiments, two or more proteins are directly linked. In some embodiments, two or more proteins as described herein are linked by a peptide linker. In various embodiments, two or more proteins as described herein are linked via a peptide linker of up to 30 amino acids. In other embodiments, two or more proteins as described herein are linked via a peptide linker of 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15 amino acids. In specific embodiments, two or more proteins as described herein are linked via a peptide linker of 10 amino acids. In specific embodiments, two or more proteins as described herein are linked via a peptide linker of 10 amino acids selected from glycine and serine.

[0097] The functional characteristics of the binding proteins as described herein fully apply to any of the multimers provided by the present invention.

[0098] Coupling sites. In some embodiments, the Her3 binding protein as described herein further comprises one or more coupling site(s) for the coupling of chemical moieties. A coupling site is capable of reacting with other chemical groups to couple the Her3 binding protein to chemical moieties. The defined number and defined position of coupling sites enables site-specific coupling of chemical moieties to the Her3 binding protein as described herein. Thus, a large number of chemical moieties can be bound to the Her3 binding protein if required. The number of coupling sites can be adjusted to the optimal number for a certain application by a person skilled in the art to adjust the amount of the chemical moieties accordingly. In selected embodiments, the coupling site may be selected from the group of one or more amino acids which can be labeled with specific chemistry such as one or more cysteine residues, one or more lysine residues, one or more tyrosine, one or more tryptophan, or one or more histidine residues. The Her3 binding protein may comprise 1 to 20 coupling site(s), preferably 1 to 6 coupling site(s), preferably 2 coupling sites, or preferably one coupling site.

[0099] Coupling domains. One embodiment provides a Her3 binding protein that comprises at least one coupling domain of 1 to 80 amino acids comprising one or more coupling sites. In some embodiments, the coupling domain of 1 to 80 amino acids may comprise alanine, proline, or serine, and as coupling site cysteine. In other embodiments, the coupling domain of 5 to 80 amino acids may consist of alanine, proline, serine, and as coupling site cysteine. In one embodiment, the coupling domain is consisting of 20 - 60 % alanine, 20 - 40 % proline, 10 - 60 % serine, and one or more cysteine as coupling site(s) at the C- or N-terminal end of the Her3 binding protein as described herein. In some embodiments the amino acids alanine, proline, and serine are randomly distributed throughout a coupling domain amino acid sequence so that no more than a maximum of 2, 3, 4, or 5 identical amino acid residues are adjacent, preferably a maximum of 3 amino acids. The composition of the 1 to 20 coupling domains can be different or identical. Chemical moieties. In some embodiments, the chemical moieties are selected from any of chelators, drugs, toxins, dyes, and small molecules. In some embodiments, at least one of the chemical moieties is a chelator designed as a complexing agent for coupling one or more further moieties to the targeted compound to the Her3 binding protein as disclosed herein. One embodiment relates to the Her3 binding protein wherein the chelator is a complexing agent for coupling one or more radioisotopes or other detectable labels.

[0100] Diagnostic moiety. In some embodiments the protein comprising an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ I D NO: 1 , wherein the binding protein has a threonine or serine at the position corresponding to position 10 of SEQ ID NO: 1 , further comprises at least one diagnostically active moiety. In various embodiments, the Her3 binding protein further comprises a diagnostic moiety. In other embodiments, the Her3 binding protein further comprises more than one diagnostic moiety. In some embodiments, such diagnostic moiety may be selected from radionuclides, chelators with radionuclide, fluorescent proteins, photosensitizers, dyes, fluorophore, enzymes, magnetic beads, metallic beads, colloidal particles, electron-dense reagent, biotin, digoxigenin, hapten, or any combination of the above. In some embodiments, a Her3 binding protein that comprises at least one diagnostic moiety can be employed, for example, as imaging agents, for example to evaluate presence of tumor cells or metastases, tumor distribution, and / or recurrence of tumor. Methods for detection or monitoring of cancer cells involve imaging methods. Such methods involve imaging Her3 related cancer cells by, for example, radio imaging or photoluminescence or fluorescence.

[0101] Therapeutic moiety. In some embodiments the Her3 binding protein comprises an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the binding protein has a threonine or serine at the position corresponding to position 10 of SEQ ID NO: 1, and further comprises at least one therapeutically active moiety. In other embodiments, the Her3 binding protein further comprises more than one therapeutically active moiety. In some embodiments, such therapeutically active moiety may be selected from a monoclonal antibody or a fragment thereof, an extracellular domain of a receptor or fragments thereof, a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or derivatives thereof, or any combination of the above. In some embodiments, the Her3 binding protein that comprises a therapeutically active component may be used in targeted delivery of any of the above listed components to the Her3 expressing tumor cell and accumulate therein, thereby resulting in low levels of toxicity to normal cells.

[0102] Radionuclides. Suitable radionuclides for applications in imaging (for example, in vitro) or for radiotherapy include for example but are not limited to the group of gamma-emitting isotopes, the group of positron emitters, the group of beta-emitters, and the group of alpha-emitters. In some embodiments, suitable conjugation partners include chelators such as 1 ,4,7,10- tetraazacyclododecane-1 ,4,7,10-tetraacetic acid (DOTA) or diethylene triamine pentaacetic acid (DTPA) or octadentate chelator (DFO) or their activated derivatives, nanoparticles and liposomes. In various embodiments, DOTA may be suitable as complexing agent for radioisotopes and other agents for imaging.

[0103] Moiety modulating pharmacokinetics. In some embodiments the Her3 binding protein comprising an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the binding protein has a threonine or serine at the position corresponding to position 10 of SEQ ID NO: 1 , further comprises at least one moiety modulating pharmacokinetics. In some embodiments, the moiety modulating pharmacokinetic is selected from an albumin-binding protein, an albumin binding small molecule, a polyethylene glycol, a human serum albumin, an immunoglobulin binding peptide or an immunoglobulin or immunoglobulin fragments, a sialic acid, or a transferrin, a transferrin receptor binding protein, a polysaccharide (for example, hydroxylethyl starch), or an unstructured amino acid sequence which increases the hydrodynamic radius such as a multimer comprising amino acids alanine, glycine, serine, proline. In various embodiments, said moiety increases the half-life of the Her3 binding protein at least 1.5-fold. Several techniques for producing Her3 binding protein with extended half-life are known in the art, for example, direct fusions of the moiety modulating pharmacokinetics with the Her3 binding protein as described above or chemical coupling methods. The moiety modulating pharmacokinetics can be attached for example at one or several sites of the Her3 binding protein through a peptide linker sequence or through a coupling site as described above.

[0104] Conjugation of proteinaceous or non-proteinaceous moieties to the Her3 binding protein may be performed applying chemical methods well-known in the art. In some embodiments, coupling chemistry specific for derivatization of cysteine or lysine residues may be applicable. Chemical coupling can be performed by chemistry well known to someone skilled in the art, including but not limited to, substitution, addition or cycloaddition or oxidation chemistry (e.g. disulfide formation).

[0105] Molecules for purification / detection. In some embodiments, additional amino acids can extend either at the N-terminal end of the Her3 binding protein or the C-terminal end or both. Additional sequences may include for example sequences introduced e.g. for purification or detection. In one embodiment, additional amino acid sequences include one or more peptide sequences that confer an affinity to certain chromatography column materials. Typical examples for such sequences include, without being limiting, Strep-tags, oligohistidine-tags, glutathione S-transferase, maltose-binding protein, inteins, intein fragments, or the albuminbinding domain of protein G.

[0106] Compositions. Various embodiments relate to a composition comprising an amino acid sequence of at least 80 % identity to the amino acid sequence of SEQ ID NO: 1 , wherein the binding protein has a threonine or serine at the position corresponding to position 10 of SEQ ID NO: 1 and wherein the binding protein has a binding affinity for human Her3 of less than 30 nM. Various embodiments relate to a composition comprising the Her3 binding protein as defined above for use in medicine. Compositions comprising the Her3 binding protein as described above may be used for clinical applications for both diagnostic and therapeutic purposes. In particular, compositions comprising the Her3 binding protein as described above may be used for clinical applications for imaging, monitoring, and eliminating or inactivating pathological cells that express Her3.

[0107] Various embodiments relate to a diagnostic composition for the diagnosis of Her3 related cancer comprising the Her3 binding protein as defined herein and a diagnostically acceptable carrier and / or diluent. These include for example but are not limited to stabilizing agents, surface-active agents, salts, buffers, coloring agents etc. The compositions can be in the form of a liquid preparation, a lyophilisate, granules, in the form of an emulsion or a liposomal preparation.

[0108] The diagnostic composition comprising the Her3 binding protein as described herein can be used for diagnosis of Her3 related cancer, as described above.

[0109] Various embodiments relate to a pharmaceutical (e.g. therapeutical) composition for the treatment of diseases comprising the Her3 binding protein as disclosed herein, and a pharmaceutically (e.g. therapeutically) acceptable carrier and / or diluent. The pharmaceutical (e.g. therapeutical) composition optionally may contain further auxiliary agents and excipients known per se. These include for example but are not limited to stabilizing agents, surfaceactive agents, salts, buffers, coloring agents etc.

[0110] The pharmaceutical composition comprising the Her3 binding protein as defined herein can be used for treatment of diseases, as described above.

[0111] The compositions contain an effective dose of the Her3 binding protein as defined herein. The amount of protein to be administered depends on the organism, the type of disease, the age and weight of the patient and further factors known per se. Depending on the galenic preparation these compositions can be administered parenterally by injection or infusion, systemically, intraperitoneally, intramuscularly, subcutaneously, transdermally, or by other conventionally employed methods of application.

[0112] The composition can be in the form of a liquid preparation, a lyophilisate, a cream, a lotion for topical application, an aerosol, in the form of powders, granules, in the form of an emulsion or a liposomal preparation. The type of preparation depends on the type of disease, the route of administration, the severity of the disease, the patient and other factors known to those skilled in the art of medicine.

[0113] The various components of the composition may be packaged as a kit with instructions for use. Use in medicine. Various embodiments relate to the Her3 binding protein as disclosed herein for use in medicine. In one embodiment, the Her3 binding protein is used in medicine to diagnose or treat cancer associated with Her3 expression. Accordingly, disclosed herein is a method of diagnosis or treatment of Her3 related cancer. The Her3 binding proteins as disclosed herein allow selective diagnosis and treatment of Her3 related cancer cells or cancer tissues. Her3 is known to be upregulated in tumor cells, possibly resulting in uncontrolled growth of tumor cells and in the formation of metastases. In one embodiment, the Her3 binding protein is used to diagnose Her3 related cancer by applying in vitro methods.

[0114] In various embodiments, the Her3 binding protein as disclosed herein may be used for diagnosis of Her3 related cancer, optionally wherein the Her3 binding protein is conjugated to a radioactive molecule. In some embodiments, the Her3 binding protein is used in (in vitro) imaging methods with labels such as radioactive or fluorescent and can be employed to visualize Her3 on specific tissues or cells, for example, to evaluate presence of Her3 related tumor cells (Her3 positive tumor cells), Her3 related tumor distribution, recurrence of Her3 related tumor (Her3 positive tumors), and / or to evaluate the response of a patient to a therapeutic treatment.

[0115] One embodiment is a method of treating a subject having Her3 related (positive) cancer (tumor, the method of treatment comprising administering to the subject the Her3 specific binding protein as described herein, optionally conjugated to a radioactive molecule and / or a cytotoxic agent. In various embodiments, the Her3 binding protein as disclosed herein may be used for treatment of Her3 related cancer, optionally wherein the Her3 binding protein is conjugated to a cytotoxic agent and / or to a radioactive molecule or expressed on the surface of target specific CarT cells. Some embodiments relate to the use of the Her3 binding protein labelled with a suitable radioisotope or cytotoxic compound or treatment of Her3 related (positive) tumor cells, in particular to control or kill Her3 related (positive) tumor cells, for example malignant cells. In one embodiment, curative doses of radiation are selectively deliveredto Her3 related (positive) tumor cells but not to normal cells.

[0116] As further described herein, in various embodiments, a Her3 related cancer is characterized by cancer cells expressing, or overexpressing, Her3. In various embodiments, the Her3 related cancer is a solid tumor expressing, or overexpressing, Her3.

[0117] Producing Her3 binding proteins. Her3 binding proteins as described herein may be prepared by any of the many conventional and well-known techniques such as plain organic synthetic strategies, solid phase-assisted synthesis techniques, fragment ligation techniques or by commercially available automated synthesizers. On the other hand, they may also be prepared by conventional recombinant techniques alone or in combination with conventional synthetic techniques. Furthermore, they may also be prepared by cell-free in vitro transcription / translation. Various embodiments relate to a polynucleotide encoding a Her3 binding protein as disclosed herein. One embodiment further provides an expression vector comprising said polynucleotide, and a host cell comprising said isolated polynucleotide or the expression vector.

[0118] Various embodiments relate to a method of producing a Her3 binding protein as disclosed herein comprising the steps of a) culturing of a host cell under suitable conditions which allow expression of said protein and b) isolating said protein.

[0119] For example, one or more polynucleotides which encode for the Her3 binding protein may be expressed in a suitable host and the produced Her3 binding protein can be isolated. A host cell comprises said nucleic acid molecule or vector. Suitable host cells include prokaryotes or eukaryotes. A vector means any molecule or entity (e.g., nucleic acid, plasmid, bacteriophage or virus) that can be used to transfer protein coding information into a host cell. Various cell culture systems, for example but not limited to mammalian, yeast, plant, or insect, can also be employed to express recombinant proteins. Suitable conditions for culturing prokaryotic or eukaryotic host cells are well known to the person skilled in the art. Cultivation of cells and protein expression for the purpose of protein production can be performed at any scale, starting from small volume shaker flasks to large fermenters, applying technologies well-known to any skilled in the art.

[0120] One embodiment is directed to a method of producing a protein as detailed above, said method comprising the following steps: (a) preparing a nucleic acid encoding a Her3 binding protein as defined herein; (b) introducing said nucleic acid into an expression vector; (c) introducing said expression vector into a host cell; (d) cultivating the host cell; (e) subjecting the host cell to culturing conditions under which a Her3 binding protein is expressed, thereby producing a Her3 binding protein as defined herein; (f) optionally isolating the Her3 binding protein produced in step (e); and (g) optionally conjugating the Her3 binding protein with further functional moieties as defined herein.

[0121] In general, isolation of purified Her3 binding protein from the cultivation mixture can be performed applying conventional methods and technologies well known in the art, such as centrifugation, precipitation, flocculation, different embodiments of chromatography, filtration, dialysis, concentration and combinations thereof, and others. Chromatographic methods are well-known in the art and comprise without limitation ion exchange chromatography, gel filtration chromatography (size exclusion chromatography), hydrophobic interaction chromatography or affinity chromatography.

[0122] For simplified purification, the Her3 binding protein can be fused to other peptide sequences having an increased affinity to separation materials. Preferably, such fusions are selected that do not have a detrimental effect on the functionality of the Her3 binding protein or can be separated after the purification due to the introduction of specific protease cleavage sites. Such methods are also known to those skilled in the art. Methods to detect Her3 in a sample. Some embodiments relate to detect Her3 in a sample, the method comprising detecting the binding of Her3 with a Her3 binding protein as described above in the sample by contacting the sample with a Her3 binding protein as described above. Some embodiments, the protein as described herein is used in methods to determine the presence of Her3. Some embodiments relate to a method of analyzing the presence of Her3 in a sample, the method comprising the following steps: (i) providing a sample that contains Her3, (ii) providing the binding protein for Her3, (iii) contacting the sample that contains Her3 with the binding protein for Her3 as described herein under conditions that permit binding of the at least one protein to Her3, (iv) isolating (eluting) the complex of a Her3 and the binding protein for Her3, and (v) determining the amount of the binding protein for Her3 which indicates the amount of Her3 in the sample of (i). In some embodiments, the sample may be a liquid sample, such as a blood sample or a urine sample or a tumor sample in liquid.

[0123] EXAMPLES

[0124] The following Examples are provided for further illustration of the invention. The invention, however, is not limited thereto, and the following Examples merely show the practicability of the invention on the basis of the above description.

[0125] Example 1. Expression and purification of Her3 binding proteins

[0126] The genes for the binding proteins were cloned into an expression vector using standard methods known to a skilled person, purified and analyzed as described below. All proteins were expressed and highly purified by affinity chromatography and gel filtration. After affinity chromatography purification by a size exclusion chromatography (SEC) was performed using an Akta system and a Superdex™ 75 HiLoad 16 / 600 column (Cytiva Life Sciences). The column had a volume of 120 ml and was equilibrated with 2 CV (PBS). The samples were applied with a flow rate of 1 ml / min using PBS as running buffer. Fraction collection started as the signal intensity reached 10 mAU. Following SDS-PAGE analysis positive fractions were pooled and their protein concentrations were measured.

[0127] Purity was confirmed by SDS-PAGE, SE-HPLC and RP-HPLC. The purity was at least 95 % as determined by SDS-PAGE. Protein concentrations were determined by absorbance measurement at 280 nm using the specific molar absorbent coefficient. RP chromatography (RP HPLC) was performed using a Vanquish Core HPLC system (Thermo Fisher Scientific) and a PLRP-S (5 pm, 300 A) column (Agilent)), with a gradient from 30 % acetonitrile containing 0.1 % TFA to 50 % acetonitrile, 0.1 % TFA.

[0128] Example 2: Mammalian expression and purification of Her3-ECD-Fc Expi293-F-cells were cultured with 0.5 - 1 Mio cells / ml in Expi293-F expression medium (Fisher Scientific, 13489756) in shake flasks at 135 rpm, 37 °C, 8 % CO2 and 95 % humidity. 1 day before transfection, cells were seeded with a density of 2.0 Mio cells / ml. On the day of transfection, cells were seeded with a density of 2.5 Mio cells / ml. 1 pg plasmid-DNA carrying the gene for the extracellular domain (ECD) of human Her3 in fusion with IgG-Fc per ml of culture volume was diluted in Opti-MEM I Reduced Serum Medium (Life Technologies, 31985- 062). ExpiFectamine was diluted in Opti-MEM I Reduced Serum Medium, according to manufacturer information and incubated for 5 min at rt. Subsequently, DNA-solution was added to the ExpiFectamine-mixture, incubated for 20 min at rt and mixed with the cells. After 16 h enhancer was added to the cells. Supernatant was collected after 96 -120 h and centrifuged. Then the pH was adjusted to pH 7.0 and the supernatant was filtered through a 0.45 pm membrane. The protein was purified via an Akta system, first by affinity chromatography with Protein A columns (Cytiva Life Sciences) and subsequently with a Superdex™ 200 HiLoad 26 / 600 column (Cytiva Life Sciences). The column had a volume of 320 ml and was equilibrated with 1.5 CV. The samples were applied with a flow rate of 2.6 ml / min using PBS as running buffer. Following SDS-PAGE analysis positive fractions were pooled and their protein concentrations were measured.

[0129] Purity was confirmed by SDS-PAGE and SE-HPLC. The purity was at least 98 % as determined by SE-HPLC. This was performed on a Ultimate Mate3000 HPLC system (Thermo Fisher Scientific). Protein concentration was determined by absorbance measurement at 280 nm using the specific molar absorption coefficient.

[0130] Example 3. Functional characterization: Biochemical binding analysis (Surface Plasmon Resonance, SPR)

[0131] Recombinant protein A was immobilized on a High Capacity Amine sensor chip (Bruker) after NHS / EDC activation resulting in approx. 1500 RU with a Sierra SPR-32 system (Bruker). The chip was equilibrated with SPR running buffer (PBS 0.05 %, Tween pH 7.3). Injection of ethanolamine after recombinant protein A immobilization was used to block unreacted NHS groups. The Fc-tagged extracellular domain of hHer3 (ECD-Fc hHer3) was injected with 90 nM followed by the injection of Her3 binding proteins with a flow rate of 30 pl / min. A flow cell without ECD-Fc hHer3 was used as reference. Upon Her3 binding proteins binding, protein analyte was accumulated on the surface increasing the refractive index. This change in the refractive index was measured in real time and plotted as response or resonance units (RU) versus time. The analytes were applied to the chip in serial dilutions. The association was performed for 120 seconds and the dissociation for 180 seconds. After each run, the chip surface was regenerated with 30 pl regeneration buffer (10 mM glycine pH 1.5) and equilibrated with running buffer. Binding studies were carried out by the use of the Sierra SPR- 32 system (Bruker) and data evaluation was operated via the Sierra Analyser software, provided by the manufacturer.

[0132] Table 1 (see below, Example 4) shows the binding affinity to human Her3 as determined by SPR. The binding affinity for hHer3 is shown as KD value (in nM); all binding proteins show specific binding for hHer3-Fc below 30 nM, hence being strong binders. The binding to human Her2 was not detectable for binding proteins shown in Table 1 .

[0133] Example 4. Functional characterization: Specific binding to cell expressed hHer3 (Flow Cytometry)

[0134] Flow cytometry was used to analyze the specific interaction of Her3 binding proteins to Her3 expressed on cells. Transfected HEK293-hHer3-cells, empty vector control HEK293-pEntry- cells were trypsinized and resuspended in medium containing FCS and washed in pre-cooled FACS blocking buffer (3% FCS; 0.1 % NaAc; PBS). A cell concentration of 1 mio cells / ml was prepared for cell staining and 100 pl / well were filled into a 96 well plate (Greiner) in triplicate for each cell line. A dilution series of proteins or 1 pg / ml Patritumab as positive control was added to Her3 expressing cells and control cells. After 45 min the supernatants were removed, and 100 pl / well rabbit anti-Strep-Tag antibody (GenScript; A00626), 1:300 diluted in FACS blocking buffer, were added. After removal of the anti-Strep-Tag antibody, goat anti-rabbit IgG Alexa Fluor 488 antibody (Invitrogen; A11008) was applied in a 1 :1000 dilution. Patritumab was detected with anti-human-IgG-Alexa 488 (Invitrogen; A-11013) with a dilution of 1 :1000. Flow cytometry measurement was conducted on the Guava easyCyte 5HT device (Merck- Millipore) at excitation wavelength 488 nm and emission wavelength 520 nm.

[0135] All tested Affilin proteins showed specific binding on human Her3 overexpressing HEK293- cells (see Table 1 below; for Her3 cell binding: strong binding: +++; medium binding: ++; low binding: +). No binding could be detected on HEK293-pEntry-cells (data not shown).

[0136] Table 1 : Specific binding to human Her3, as determined by SPR and by cell binding

[0137] Example 5. Functional characterization: Specific binding to hHer3 - KD-determination (Flow cytometry) Her3 overexpressing HEK293-cells were trypsinized, washed with FACS blocking buffer, seeded in 96-well round bottom plates with a density of 0.1 mio cells / 100 l / well and protein dilution series were added to the cells. After 45 min the supernatants were removed, and cells were washed. Binding was detected with 100 l / well rabbit anti-Strep-Tag antibody 1 :300 diluted in FACS blocking buffer in a first step and goat anti-rabbit IgG Alexa Fluor 488 antibody in a 1 :1000 dilution in a second step. The read out was described above. The binding affinity for hHer3 (KD) was below 1 nM (see Table 2).

[0138] Table 2. Specific binding to human Her3 on Her3 overexpressing cells Example 6. Functional characterization: High affinity binding to hHer3 in serum after 24 h incubation (ELISA)

[0139] High binding plates (Greiner, 781061) were coated with 2.5 pg / ml recombinant human Her3- ECD-Fc over night at 4 °C. Dilution series of test compounds in 100 % mouse serum or 100 % human serum for 24 h at 37 °C. ELISA-plates were washed 3 times with PBST (PBS, 0.1 % Tween) and blocked with 3 % BSA / 0.5 % Tween / PBS 2 h at RT. Dilution series after 0 h or 24 h incubation in the presence of serum were incubated on ELISA-plates for 1 h at rt. After washing with PBST, wells were incubated with biotinylated anti-ubiquitin-antibody (1 :300) for 1 h at rt. The binding was visualized with Streptavidin-HRP (1 :5.000). Her3 binding proteins are stable in human and mouse serum. No significant change in KD after 24 h serum incubation could be detected (Table 3). Figure 1 shows the KD of 229627 before and after 24 h incubation in mouse or human serum. For Affil in-226967, a KD of 0.7- 1.0 nM was calculated without loss of binding in mouse or human serum after 24 h.

[0140] Table 3. High affinity binding to hHer3 in serum after 24 h incubation at 37 °C

[0141] Appendix - SEQUENCES SEQ ID Her3 amino acid sequence

[0142] NO: binding protein

[0143] 1 226967 MQIFVKTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I EKDSVLALLLN LRAA

[0144] 2 226969 MQI FVKTLTSN EQYKGKTITLEVEPSDTI EN VKAKIQDKEGI PPDQQRLI WAGKQLEDGRTLSDYNISDESILALLLNLRAA

[0145] 3 226971 MQIFVKTLTTQVSPPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I RAH DQLALLLELRAA

[0146] 4 228269 MQIFVKTLTTKYDIPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYNIVQNSMLALLLNLRAA

[0147] 5 229625 MQIFVKTLTTKYDIPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I VQNSMLALLLNLRAAGGGGSGGGGSMQIF VKTLTTKYDIPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGK QLEDGRTLSDYN I VQNSM LALLLN LRAA

[0148] 6 229627 MQIFVKTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I EKDSVLALLLNLRAAGGGGSGGGGSMQIF VKTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGK QLEDGRTLSDYNIEKDSVLALLLNLRAA

[0149] 7 229629 MQI FVKTLTSN EQYKGKTITLEVEPSDTI EN VKAKIQDKEGI PPDQQRLI WAGKQLEDGRTLSDYNISDESILALLLNLRAAGGGGSGGGGSMQIF VKTLTSNEQYKGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIWAGK QLEDGRTLSDYNISDESILALLLNLRAA

[0150] 8 229717 MQIFVVTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGRQLEDGRTLSDYNIEKDSVLALLLNLRAA

[0151] 9 229736 MQIFVVTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I EKDSVLALLLN LRAA

[0152] 10 230501 MQI FVVTLTSN EQYKGKTITLEVEPSDTI EN VKAKIQDKEGI PPDQQRLI WAGKQLEDGRTLSDYNISDESILALLLNLRAA

[0153] 11 230502 MQIFVVTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGYQLEDGRTLSDYN I EKDSVLALLLN LRAA

[0154] 12 230521 MQIFVKTLTTTITPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN ITADAM LALLLN LRAA

[0155] 13 230572 MQIFVKTLTSRPPLKGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I PN KSM LALLLH LRAA

[0156] 14 230588 MQIFVQTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPYQQRLI WAGKQLEDGRTLSDYN I EKDSVLALLLN LRAA

[0157] 15 230593 MQIFVKALTTQVSPPGKTITLEVEPSDTIEKVKAKIQDKEGI PPDQQRLI WAGKQLEDGRTLSDYN I RAH DQLALLLELRAA

[0158] 16 230595 MQIFVRTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I EKDSVLALLLN LRAA

[0159] 17 230601 MQIFVKTLTTQVSPPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGIQLEDGRTLSGYNIEKDGVLALLLNLRAA

[0160] 18 230603 MQIFVKTLTTQVSPPGKTITLEVEPSDTIENVKAKIQDKEGIPSDQQRLI WAGKQLEDGRTLSDYNIVQNSMLALLLNLRAA

[0161] 19 230616 MQIFVKALTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I EKDSVLALLLN LRAA

[0162] 20 230627 MQIFVVTLTTTIAPVGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI YAGKQLEDGRTLSDYNIEKDSVLALLLNLRAA

[0163] 21 230787 MQIFVKTLTTQVSPPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I KH NEM LALLLSLRAA

[0164] 22 230788 MQIFVKTLTTQISPPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I DNGAH LALLLN LRAA

[0165] 23 230789 MQIFVKTLTTQISPPGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYN I PANGVLALLLSLRAA 230791 MQIFVKTLTTHYSQYGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLI WAGKQLEDGRTLSDYNINQNSTLALLLNLRAA

[0166] 230795 MQIFVKTLTTQISPTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQKLW WAGKQLEDGRTLSDYNIYPHSFLGLTLRAA

[0167] 230796 MQIFVKTLTSLPPHSGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLIW AGKQLEDGRTLSDYNIPTQGVLALLLRAA

[0168] 230801 MQIFVKTLTSHPRIGGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLVW AGKQLEDGRTLSDYNITSHAMLALLLSLDLRAA

[0169] 230802 MQIFVKTLTTTIAPVGKTITLEVEPSDTIENVKAKIRDKEGIPPDQQRLI

[0170] WAGKQLEDGRTLSDYNIEKDSVLALLLNLRAA

[0171] 230804 MQIFVKTLTTTVAPVGKTITLEVEPSDTIGYVKAKIQDKEGIPPDQQRLI

[0172] WAGKQLEDGRTLSDYNIEKDSVLALLLNLRAA

[0173] 230808 MQIFVKTLTSFDPWHGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLV WAGKQLEDGRTLSDYNIQHFDNLALLLNLRAA

[0174] 230809 MQIFVKTLTSLQPHTGKTITLEVEPSDTIENVKAKIQDKEGIPPDQQRLV

[0175] WAGKQLEDGRTLSDYNIVENDYLALLLQLRAA

[0176] ***

Claims

CLAIMS1. A binding protein for human Her3 comprising(a) an amino acid sequence with at least 80 % identity to SEQ ID NO: 1 , wherein the amino acid corresponding to position 10 of SEQ ID NO: 1 is threonine (T) or serine (S), and(b) wherein the Her3 binding protein has a binding affinity for human Her3 of less than 30 nM, preferably less than 10 nM, preferably less than 6 nM, as determined by Surface Plasmon Resonance (SPR).

2. The binding protein for human Her3 according to claim 1 wherein the amino acid in position 74 of SEQ ID NO: 1 is Alanine (A).

3. The binding protein for human Her3 according to claim 1 or claim 2 wherein and the amino acid in position 76 of SEQ ID NO: 1 is Leucine (L).

4. The binding protein for human Her3 according to claims 1-3, comprising an amino acid sequence selected from any of SEQ ID NOs: 1-31.

5. A multimer comprising the binding protein for human Her3 according to claims 1-4 wherein the multimer is a dimer, a trimer, a tetramer, a pentamer, or a hexamer, preferably a dimer.

6. The binding protein according to claims 1-5, wherein the binding protein comprises additionally at least one molecule modulating pharmacokinetics selected optionally from an albumin-binding protein, an albumin binding small molecule, a serum albumin, an immunoglobulin binding protein, an immunoglobulin or immunoglobulin fragment, a polysaccharide, an unstructured amino acid sequence comprising amino acids alanine, glycine, serine, proline, a polyethylene glycol, a sialic acid, a transferrin, a fatty acid, and a transferrin receptor binding protein, or any combination thereof.

7. The binding protein according to claims 1-6, wherein the binding protein comprises additionally at least one diagnostically active moiety, optionally selected from a radionuclide, a chelator with a radionuclide, fluorescent protein, photosensitizer, dye, or enzyme, or any combination of the above,8. The binding protein according to claims 1-6, wherein the binding protein comprises additionally at least one therapeutically active moiety, optionally selected from a monoclonal antibody or a fragment thereof, a binding protein, a receptor or receptor domain, a radionuclide, a chelator with a radionuclide, a cytotoxic compound, a cytokine, a chemokine, an enzyme, or derivatives thereof, or any combination of the above.

9. The binding protein according to claims 1-8, for use in diagnostics or treatment of cancer.

10. A nucleic acid molecule encoding the binding protein according to claims 1-8.

11. A vector comprising the nucleic acid molecule of claim 10.

12. A host cell or a non-human host comprising the binding protein according to claims 1- 8, a nucleic acid as defined in claim 10, and / or a vector of claim 11.

13. A composition comprising the binding protein according to claims 1-8 for use in medicine, preferably for use in the diagnosis or treatment of Her3 positive tumors or cancer cells.

14. A method of producing the binding protein according to claims 1-8 comprising a) culturing a host cell of claim 12 under suitable conditions in order to obtain said binding protein and b) isolating said binding protein and optionally isolating said binding protein from the host cell of claim 12 and / or the medium in which the host cell of claim 12 is growing.

15. A method of detecting Her3 in a sample, the method comprising (a) providing a sample, (b) providing the binding protein according to claims 1-8, (c) contacting the binding protein with the sample under conditions that permit binding of the binding protein according to claims 1-8; and (d) determine the binding to Her3 in the sample.

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