HER3 binding polypeptides
09745350 · 2017-08-29
Assignee
Inventors
Cpc classification
A61K51/088
HUMAN NECESSITIES
G01N33/57492
PHYSICS
C07K14/00
CHEMISTRY; METALLURGY
International classification
A61K51/08
HUMAN NECESSITIES
C07K14/00
CHEMISTRY; METALLURGY
Abstract
The present disclosure relates to polypeptides which bind to human epidermal growth factor receptor 3 (HER3) and to use of such polypeptides in imaging and therapy. The disclosure provides an HER3 binding polypeptide comprising a HER3 binding motif, which motif consists of the amino acid sequence EKYX.sub.4AYX.sub.7EIW X.sub.11LPNLTX.sub.17X.sub.18QX.sub.20 AAFIGX.sub.26 LX.sub.28D (SEQ ID NO:110).
Claims
1. A HER3 binding polypeptide, comprising a HER3 binding motif (BM), which motif consists of an amino acid sequence selected from SEQ ID NO. 1-35.
2. The HER3 binding polypeptide according to claim 1, selected from SEQ ID NO:71-105.
3. The HER3 binding polypeptide according to claim 1, wherein the off-rate (k.sub.off) of the interaction between said HER3 binding polypeptide and human HER3 is at least four-fold reduced, when compared to the off-rate (k.sub.off) of the interaction between a comparative HER3 binding polypeptide comprising the amino acid sequence SEQ ID NO:107 and human HER3, as measured using the same experimental conditions.
4. The HER3 binding polypeptide according to claim 1, wherein the K.sub.D value of the interaction between said HER3 binding polypeptide and human HER3 is at most 1×10.sup.−8 M.
5. The HER3 binding polypeptide according to claim 1, further comprising a second moiety consisting of a polypeptide having a biological activity.
6. The HER3 binding polypeptide according to claim 5, wherein said biological activity is selected from a group consisting of a therapeutic activity, a binding activity and an enzymatic activity.
7. The HER3 binding polypeptide according to claim 1, further comprising a label, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.
8. The HER3 binding polypeptide of claim 7, further comprising a second moiety comprising a polypeptide having a biological activity.
9. A composition comprising a HER3 binding polypeptide according to claim 1 and at least one excipient or carrier.
10. The composition of claim 9, wherein the HER3 binding polypeptide further comprises a second moiety comprising a label or a polypeptide having a biological activity.
11. The composition of claim 10, wherein the second moiety comprises a label.
12. The composition of claim 10, wherein the second moiety comprises a polypeptide having a biological activity.
13. A polynucleotide encoding an HER3 binding polypeptide according to claim 1.
14. The polynucleotide of claim 13, wherein the HER3 binding polypeptide further comprises a second moiety comprising a polypeptide having a biological activity.
15. A method of detecting HER3, comprising contacting a sample suspected to contain HER3 with a HER3 binding polypeptide according to claim 1, and detecting the binding of the HER3 binding polypeptide to indicate the presence of HER3 in the sample.
16. The method of claim 15, wherein the HER3 binding polypeptide further comprises a second moiety comprising a label or a polypeptide having a biological activity.
17. The method of claim 16, wherein the second moiety is the polypeptide having a biological activity.
18. The method of claim 16, wherein the second moiety is the label.
19. A method of in vivo imaging of the body of a subject having or suspected of having a cancer characterized by over expression of HER3, comprising the steps of: administering a radiolabeled HER3 binding polypeptide according to claim 7, wherein the radionuclide is suitable for imaging, into the body of the mammalian subject; and obtaining one or more images, within 1-72 hours of administration of the radiolabeled polypeptide, of at least a part of the subject's body using a medical imaging instrument, said image(s) indicating the presence of the radionuclide inside the body.
20. The method of claim 19, wherein the radiolabeled HER3 binding polypeptide further comprises a second moiety comprising a polypeptide having a biological activity.
21. A method of treatment of a HER3-expressing cancer, comprising administering to a subject in need thereof an effective amount of a HER3 binding polypeptide of claim 1.
22. The method of claim 21, wherein the HER3 binding polypeptide further comprises a second moiety comprising a label or a polypeptide having a biological activity.
23. The method of claim 22, wherein the second moiety is the polypeptide having a biological activity.
24. The method of claim 22, wherein the second moiety is the label.
Description
BRIEF DESCRIPTION OF THE FIGURES
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(12) The invention will now be illustrated further through the non-limiting description of experiments conducted in accordance therewith. Unless otherwise specified, conventional chemistry and molecular biology methods were used throughout.
EXAMPLES
Example 1
Rational Design of a New Generation of HER3 Binding Z Variants
(13) In this Example, an affinity maturation library was constructed based on HER3 binding polypeptides Z05416 (SEQ ID NO:106) and Z05417 (SEQ ID NO:107), selected from a previous affinity maturation library (Kronqvist et al, 2010, supra), as well as on results from an alanine scanning analysis of Z05416 assessed by Fluorescence-Activated Cell Sorting (FACS) as described in this Example.
(14) Materials and Methods
(15) Labeling of HER3 and HSA:
(16) Biotinylation of recombinant human HER3/Fc chimera (R&D Systems, cat. no. 348-RB-050), here denoted HER3-Fc, was performed using the Biotin-XX Microscale Protein Labeling Kit (Invitrogen, cat. no. B30010) according to the supplier's recommendations. The concentration of labeled protein was determined using amino acid analysis. The extracellular domain of HER3 (Sino Biological Inc., cat. no. 10201-H08H), here denoted HER3-ECD, was conjugated with biotin carboxylic acid, succinimidyl ester in NaHCO.sub.3 (0.1 M, pH 8.5) for 1.5 h. Subsequently, glycine was added to stop the reaction followed by buffer exchange to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) using a PD MiniTrap G-25 column (GE Healthcare, cat. no. 28-9180-07) according to manufacturer's recommendations. Human serum albumin (HSA; Sigma, cat. no. A-3782) was fluorescently labeled using ALEXA FLUOR 647 (Fluorescent Dye)succinimidyl ester (Invitrogen, cat. no. A20006) according to the supplier's recommendations. The protein buffer was changed to PBS (10 mM phosphate, 137 mM NaCl, 2.68 mM KCl, pH 7.4) or PBS supplemented with 0.1% PLURONIC F108 NF Surfactant (PBSP; BASF Corporation, cat. no. 30085231) in order to remove any excess fluorophore.
(17) Alanine Scanning Mutagenesis:
(18) The Z variant Z05416, selected previously as described in Kronqvist et al (2010, supra), was used as a template for construction of 12 mutants in which residues at positions 9, 10, 11, 13, 14, 17, 18, 24, 25, 27, 32 and 35 were replaced with an alanine, one for each of the mutants. Conventional site-directed mutagenesis was performed using a vector (pSCZ05416 (Kronqvist et al, 2010 supra)) encoding Z05416, and oligonucleotides encoding the respective alanine replacements (using the codon GCG). In an additional mutant, the original alanine residue at position 28 within Z05416 was substituted to a valine (using the codon GTG) by the same means. Gene sequences were digested with NheI and XhoI restriction enzymes (New England Biolabs) and ligated to the staphylococcal display vector pSCZ1 (Kronqvist et al, Protein Engineering Design & Selection 21: 247-255 (2008)) that had been digested with the same enzymes, using T4 DNA ligase (New England Biolabs) according to supplier's recommendations. The E. coli strain RR1ΔM15 (Rüther, Nucleic Acids Res 10:5765-5772 (1982)) was used as host for plasmid construction and preparation was performed with a JETSTAR Kit (Genomed, cat. no. 220 020) according to the supplier's recommendations. BigDye Thermo Cycle Sequencing reactions and an ABI Prism 3700 instrument (Applied Biosystems, Foster City, Calif.) were used to verify the presence of the alanine or valine mutation in each plasmid. The constructs were transformed to electrocompetent S. carnosus TM300, as described in Lofblom et al (J Appl Microbiol 102: 736-747 (2007)).
(19) FACS Analysis of Alanine and Valine Mutants:
(20) Staphylococcal cells displaying the alanine and valine mutants were inoculated into 10 ml tryptic soy broth supplemented with yeast extract (TSB-YE; Merck, Darmstadt, Germany) and with 20 μg/ml chloramphenicol, and grown overnight at 37° C. at 150 rpm agitation. 10.sup.6 cells from overnight cultures were washed with 800 μl PBS supplemented with 0.1% PLURONIC F108 NF Surfactant (PBSP; pH 7.4; BASF Corporation, cat. no. 30085231). The cells were pelleted by centrifugation (3500×g, 4° C., 6 min) and resuspended in 50 μl of PBSP containing biotinylated 5 nM HER3-Fc. Equilibrium binding was reached by incubation at room temperature for 2 h with gentle mixing. The cells were washed with 180 μl ice-cold PBSP, followed by incubation for 40 min in the dark in 200 μl ice-cold PBSP containing 150 nM Alexa Fluor® 647-conjugated HSA and Streptavidin-Alexa 488 conjugate. Following one wash with 180 μl ice-cold PBSP, cells were resuspended in 200 μl ice-cold PBSP prior to flow cytometric analysis. The mean fluorescence intensity (MFI) was measured using a FACS Vantage SE (BD Biosciences, San Jose, Calif.) flow cytometer. The experiment was carried out in duplicates on different days using freshly prepared solutions.
(21) Library Design:
(22) A new library was designed, in which 13 positions in the Z molecule were biased towards the amino acid residues based on the sequences of the HER3 binding Z variants Z05416 and Z05417 (Kronqvist et al, 2010 supra). Each position was randomized with 17 codons corresponding to amino acids: A, E, F, G, H, I, K, L, M, N, Q, R, S, T, Y, V, W (excluding C, D, P in all positions) with the amino acid residues based on the sequences of the HER3 binding Z variants Z05416 and Z05417 spiked in at a higher proportion to generate an average mutation frequency of approximately three mutations per molecule (Table 1). The randomization frequency in each position was also normalized with the results from the alanine scanning experiment described above, resulting in less mutations in important positions and vice versa (Table 1).
(23) A SLONOMAX library of double-stranded DNA encoding partially randomized positions in helix 1 and 2 of the HER3 binding polypeptide, flanked with the restriction sites XhoI and SacI (5′-CTC GAG GCG GAA GCC AAA TAC GCC AAA GAA NNN NNN NNN GCG NNN NNN GAG ATC NNN NNN TTA CCT AAC TTA ACC NNN NNN CAA NNN NNN GCC TTC ATC NNN AAA TTA NNN GAT GAC CCA AGC CAG AGC TCT C′ (SEQ ID NO:108; randomized codons illustrated as NNN) was ordered from Sloning Biotechnology GmbH (Pucheim, Germany). The theoretical distributions of amino acid residues in the new library for the 13 variable Z positions are given in Table 1.
(24) TABLE-US-00001 TABLE 1 Library design No of Randomization No of Amino acid position in amino (amino acid amino the Z variant molecule Randomization (amino acid abbreviations) acids Proportion abbreviations) acids Proportion 9 A, E, F, G, H, I, L, M, N, Q, S, T, V, W, Y 15 0.8 K, R 2 44.00 10 A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W 16 0.2 Y 1 96.00 11 A, E, F, G, , H, I, K, L, M, N, Q, R, V, W, Y 15 1.9 T, S 2 35.75 13 A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W 16 1.8 Y 1 96.80 14 A, E, G, H, I, K, L, M, N, Q, R, S, T, V, W 15 1.9 F, Y 2 71.20 17 A, E, F, G, H, I, K, L, M, N, Q, R, S, T, V, Y 16 0.2 W 1 71.20 18 A, E, F, G, H, I, K, L, M, N, R, S, T, V, W, Y 16 1.8 Q 1 71.20 24 A, E, F, G, H, I, K, L, M, N, Q, R, S, T, W, Y 16 1.8 V 1 71.20 25 A, E, F, G, H, I, K, L, M, N, Q, S, T, V, W, Y 16 1.8 R 1 71.20 27 A, E, F, G, H, I, L, M, N, Q, R, S, T, V, W, Y 16 1.8 K 1 71.20 28 E, F, G, H, I, K, L, M, N, Q, R, S, T, V, W, Y 16 1.8 A 1 71.20 32 A, E, F, H, I, K, L, M, N, Q, R, T, V, W, Y 15 1.9 S, G 2 35.75 35 A, E, F, G, H, I, K, L, M, N, R, S, T, V, W, Y 16 1.8 Q 1 71.20
(25) Library Construction and Cloning:
(26) The library was amplified using Phusion DNA polymerase (Finnzymes, cat. no. F530L) during 11 cycles of PCR. The PCR product was purified using QIAquick PCR Purification Kit (Qiagen, cat. no. 28106) according to the supplier's recommendations. Subsequently, the library oligonucleotides were digested by XhoI and SacI-HF (New England Biolabs) restriction enzymes and purified by preparative gel electrophoresis (2% agarose gel) using QIAquick gel extraction kit (Qiagen, cat.no. 28704). A modified version of the S. carnosus expression vector pSCZ1 (Kronqvist et al, 2008 supra) was restricted by XhoI and SacI-HF enzymes and purified by preparative gel electrophoresis as described above. The library oligonucleotides were ligated into the vector using T4 DNA ligase at a 1:5 molar ratio of vector to insert, followed by phenol-chloroform extraction and ethanol precipitation for purification and concentration of DNA fragments. Next, the library-encoding plasmids were transformed into electrocompetent E. coli SS320 (Lucigen, cat. no. 60512-1) by electroporation and individual clones were sequenced for library validation by BigDye Thermo Cycle Sequencing using an ABI Prism 3700 instrument (Applied Biosystems, Foster City, Calif.). The library plasmids were subsequently isolated using a JETSTAR Maxi Kit (Genomed cat. no. 220020), purified by phenol-chloroform extraction and concentrated by isopropanol precipitation. Finally, the library (hereafter denoted Sc:Z.sub.HER3LIB2) was transformed by electroporation into electrocompetent S. carnosus as previously described (Lofblom et al, 2007, supra).
(27) Library quality analysis: An aliquot of Sc:Z.sub.HER3LIB2 (at least ten times the library size, i.e. more than 6.7×10.sup.8) was inoculated to 100 ml TSB-YE with 20 μg/ml chloramphenicol and grown overnight at 37° C. and 150 rpm. After 16 h, 10.sup.7 cells were washed once with 1 ml PBSP. The cells were pelleted by centrifugation (3500×g, 4° C., 6 min) and resuspended in PBSP containing 225 nM ALEXA FLUOR 647-conjugated HSA and incubated for 1 h at RT in the dark. Following one wash with 1 ml ice-cold PBSP, cells were resuspended in 300 μl ice-cold PBSP prior to flow cytometric analysis. The mean fluorescence intensity (MFI) was measured using a MoFlo Astrios Cell Sorter (Beckman Coulter) flow cytometer.
(28) Results
(29) FACS Analysis of Alanine and Valine Mutants of a HER3 Binding Polypeptide:
(30) Alanine scanning mutagenesis was used to study the high-affinity binding site of known HER3 binder Z05416 to the extracellular domain of human HER3-Fc. The thirteen originally randomized residues in the Z scaffold were each substituted to an alanine or valine amino acid, and each construct was subcloned into the staphylococcal display vector for subsequent transformation to the staphylococcal host. Staphylococcal cells displaying the thirteen replacements were incubated with biotinylated HER3-Fc. Next, cells were washed and incubated with Streptavidin-Alexa 488 conjugate and HSA for binding to the albumin binding protein (ABP) in fusion with the Z variant molecules to monitor the surface expression level and normalization of the antigen-binding signal. After washing, the effect of each alanine or valine replacement on HER3 binding was analyzed using flow cytometry. The results showed that alanine substitutions at positions 9, 10, and 17 drastically reduced the affinity for HER3-Fc, indicating that these positions are involved in target binding (
(31) Library Construction and Cloning:
(32) The new library was designed based on the previously selected HER3 binding polypeptides Z05416 and Z05417 as well as the result from the alanine scan. The theoretical size of the designed library was 5.6×10.sup.6 clones, including Z variants with up to 3 mutations. The library of DNA fragments was cloned into the staphylococcal expression vector. Sequence analysis of individual library members verified a distribution of codons in accordance with the theoretical design and a low proportion of frame shifts (1.8%). The mutation frequency was somewhat lower than the intended 3 out of 13 amino acids; on average 2 mutations per clone were found. The library was transformed into S. carnosus generating a diversity of approximately 6.7×10.sup.7 individual clones.
(33) Library Quality Analysis:
(34) In order to verify that the Z variants of the maturation library Sc:Z.sub.HER3LIB2 were functionally displayed on the bacterial surface, staphylococcal cells from the library were incubated with fluorescently labeled HSA and analyzed using flow cytometry. The result showed that around 80% of the library expressed full-length proteins with functional ABP fusions on the cell surface. A new maturated library of HER3 binding polypeptides had thus been successfully constructed.
Example 2
Selection, Screening and Characterization of HER3 Binding Z Variants
(35) Materials and Methods
(36) Cell Labeling and Staphylococcal Cell Sorting Using FACS:
(37) An aliquot of the library Sc:Z.sub.HER3LIB2 designed in Example 1 (at least ten times the library size, i.e. more than 6.7×10.sup.8 variants) was inoculated into TSB-YE with 10 μg/ml chloramphenicol and grown overnight at 37° C. and 150 rpm. The following day, cells were harvested by centrifugation (6000 rpm, 6 min, 4° C.) and washed in PBSP before addition of HER3-ECD, biotinylated as described in Example 1. Cells were incubated at room temperature with gentle mixing until equilibrium binding was reached. Washing with ice-cold PBSP was performed prior to incubation with 5 μg/ml streptavidin conjugated with phycoerythrin (SAPE; Invitrogen, cat. no. S21388) and 300 nM ALEXA FLUOR 647-conjugated HSA for 30 min on ice in the dark. Cells were once again washed and finally resuspended in ice-cold PBSP. The library was labeled and sorted in altogether four rounds using a MOFLO Astrios (Beckman Coulter) flow cytometer. For sort 1 and 2, the library was labeled with 5 nM of biotinylated HER3-ECD while sort 3 was performed using two different labeling strategies with either 1 nM (strategy 1) or 5 nM (strategy 2) of biotinylated HER3-ECD. In strategy 2, the library was additionally subjected to an off-rate selection by subsequently incubating cells with 5 nM of non-biotinylated HER3-ECD for 1 h at room temperature prior to labeling with SAPE and HSA ALEXA FLUOR 647. Prior to sort 4, both strategies from sort 3 were subjected to an off-rate selection by first incubating the cells with 1 nM of biotinylated HER3-ECD; cells were then washed before addition of 1 nM of non-biotinylated HER3-ECD for 4 h at room temperature. For each round of sorting, a number of cells corresponding to approximately ten times the library size was analyzed in the flow cytometer and the top fraction of cells (approximately 0.1-0.5%), with the highest ratio of HER3 binding to cell surface expression, was gated out and sorted into an eppendorf tube with TSB-YE. Subsequently, sorted cells were inoculated into TSB-YE supplemented with chloramphenicol (1 μg/ml) for overnight amplification prior to the next sorting round. Finally, isolated cells after the 3.sup.rd and 4.sup.th sorting rounds were spread on agar plates containing chloramphenicol.
(38) Sequencing:
(39) Sequencing of individual staphylococcal clones was performed after sorting rounds 3 and 4: 96 individual colonies from each selection strategy were picked for BigDye Thermo Cycle Sequencing reactions using an ABI Prism 3700 instrument (Applied Biosystems, Foster City, Calif.).
(40) On-Cell Affinity Ranking:
(41) 40 individual Sc:Z.sub.HER3LIB2 clones, selected based on the sequencing result, were inoculated into TSB-YE with chloramphenicol (10 μg/ml) and grown overnight at 37° C. and 150 rpm. Cells were then pelleted by centrifugation and washed in PBSP before resuspension in either 0.5 nM or 2 nM of biotinylated HER3-ECD. After 1 h incubation at room temperature with gentle mixing, cells were washed with ice-cold PBSP and labeled with SAPE at a final concentration of 5 μg/ml and ALEXA FLUOR 647-conjugated HSA at a concentration of 300 nM for 30 min on ice. Finally, cells were washed and resuspended in ice-cold PBSP. All samples were ranked based on the ratio between mean fluorescence intensities (MFI) from HER3 binding and cell surface expression signals in a Gallios (Beckman Coulter) flow cytometer. In addition, the precursor Z variant Z05417 was analyzed for comparison.
(42) Results
(43) Flow Cytometric Sorting for Isolation of Improved Z Variants:
(44) For isolation of matured HER3 binding Z variants, the staphylococcal library was subjected to four rounds of FACS. The selection stringency was modified throughout the selection process by changing the sorting parameters and gates, and also by decreasing the target concentration as well as incorporating off-rate selections at later sorting rounds. Prior to the 3.sup.rd round of sorting, the selection scheme was divided into two different tracks using different labeling strategies (referred to as strategy 1 and strategy 2) as described in the Materials and methods section.
(45) The visualization of the target-binding properties of the library in the flow cytometer revealed an enrichment of HER3-positive clones in each sorting round (
(46) Sequencing:
(47) After the 3.sup.rd sorting round, 70 unique sequences out of 130 total reads were identified, while 37 unique sequences out of 142 reads where identified after the 4.sup.th and final round. These results indicated an enrichment of individual HER3 binding clones between sorting rounds 3 and 4.
(48) On-Cell Affinity Ranking:
(49) 40 unique clones from either the 3.sup.rd or the 4.sup.th sorting round were affinity ranked by flow cytometry, by determining the ratio between the HER3 binding signal and the surface expression level. The vast majority of analyzed clones showed improved affinity towards HER3 compared to the precursor HER3 binding polypeptide Z05417. The amino acid sequences of the 58-mer Z variants of these binders with improved affinity are listed in
Example 3
Production and Characterization of a Selection of HER3 Binding Z Variants
(50) In this Example, the top ten candidates (Z08694-Z08703; SEQ ID NO:71-80; see
(51) Materials and Methods
(52) Cloning, Protein Expression and Purification of Z Variants:
(53) DNA sequences encoding ten HER3 binding polypeptides selected in Example 2 (Z08694-Z08703; SEQ ID NO:71-80) were amplified from colonies by PCR, using primers introducing NdeI and XhoI restriction sites. The Z variants were subsequently cloned into the NdeI and XhoI restricted expression vector pET26b+ (Novagen), for encoding of monomeric Z variants with a C-terminal His.sub.6-tag in the format Z#####-LEHHHHHH (SEQ ID NO: 122). The plasmids were transformed into Rosetta(DE3) E. coli cells by heat shock. Cells were cultured in TSB-YE at 37° C. and protein expression was induced by the addition of IPTG (isopropyl-β-D-1-thiogalactopyranoside) to a final concentration of 1 mM when the OD.sub.600 had reached approximately 1. After incubation overnight at 25° C., the cells were harvested by centrifugation at 4000 rpm for 8 min at 4° C. The cell pellets were resuspended in lysis buffer (7 M guanidinium chloride, 47 mM Na.sub.2HPO.sub.4, 2.65 mM NaH.sub.2PO.sub.4, 10 mM Tris-HCl, 100 mM NaCl) and incubated for 2 h at 37° C. and 150 rpm. Subsequently, cell debris was removed by centrifugation at 16000 rpm for 20 min at 4° C. The supernatants were isolated and the Z variants were purified by IMAC using a HisPurTM Cobalt resin (Thermo Scientific, cat. no 89965) under denaturing conditions. Buffer was exchanged to PBS by dialysis using Slide-A-Lyzer dialysis cassettes, 3.5 kDa cutoff (Thermo Scientific). The molecular weight and the purity of the purified Z variants were verified by LC/MS (Agilent Technologies 6520 ESI-Q-TOF) and SDS-PAGE. The protein concentration was determined by absorbance measurement at 280 nm.
(54) Off-Rate Ranking by Biosensor Analysis:
(55) All biosensor assays were performed on a ProteOn XPR36 instrument (Bio Rad Laboratories, CA, USA) using PBS+0.05% Tween (PBST) as running buffer and 15 mM NaOH for regeneration. In all experiments, subtraction of responses from each sample over a blank surface was performed to minimize buffer contributions. Recombinant human and murine HER3-Fc (R&D systems, cat. no. 348-RB-050 and 4518-RB-050, respectively) was immobilized on separate GLC chips (Bio Rad Laboratories, CA, USA) using standard sulfo-NHS/EDAC amine coupling chemistry. The ligands were diluted to a final concentration of 10 μg/ml in 10 mM NaAc, pH 4.5, and final immobilization levels were approximately 3000 RU. The purified polypeptides were subjected to off-rate ranking by injection of 25 nM of each Z variant over the immobilized ligands at a flow rate of 100 μl/min; association and dissociation time was set to 120 and 1800 seconds, respectively. Each Z variant was analyzed in duplicate and the fold-change of the off-rates of each HER3-specific Z variant compared to the k.sub.off-value reported previously for the Z variant Z05417 (Kronqvist et al, 2010 supra) was determined by fitting a curve to the dissociation phase.
(56) Biosensor Analysis of HER3 Binding Before and after Heat Treatment:
(57) Z variants Z08698 and Z08699 at concentrations of 25 and 50 nM were subjected to heat treatment at 90° C. for 15 min. Binding to HER3 was evaluated by injection of 400 μl of each sample, before and after heat treatment, over immobilized human and mouse HER3, respectively, at a flow rate of 100 μl/min.
(58) Circular Dichroism Spectroscopy:
(59) Z variants Z08698 and Z08699 were diluted to 0.2 mg/ml in PBS and subjected to circular dichroism analysis. For each Z variant, a CD spectrum at 250-195 nm was recorded at 20° C. using a Jasco J-810 spectropolarimeter (Jasco Scandinavia AB) and a cell with an optical path-length of 1 mm. In addition, a variable temperature measurement (VTM) was performed to determine the melting temperature (Tm). In the VTM, the absorbance was measured at 220 nm while the temperature was raised from 20 to 90° C., with a temperature slope of 5° C./min.
(60) Affinity Determination by Biosensor Analysis:
(61) The equilibrium dissociation constant (K.sub.D) was determined for the Z variants Z08698 and Z08699. A GLC sensor chip (Bio Rad Laboratories, CA, USA) was immobilized with human HER3-Fc as described above, with a final immobilization level of approximately 650 RU. Duplicate injections of a two-fold dilution series of each Z variant, ranging from 7.2 to 1.8 nM for Z08698 and from 6.8 to 1.7 nM for Z08699 (protein concentrations determined by amino acid analysis), were injected over immobilized human HER3. The flow rate was set to 50 μl/min and the association and dissociation was followed for 300 s and 4 h respectively. The on-rate (k.sub.on) and off-rate (k.sub.off) as well as the K.sub.D value were determined by fitting the sensorgrams to a Langmuir one-site binding model.
(62) Results
(63) Protein Expression and Purification:
(64) The ten monomeric Z variant molecules (Z08694-Z08703) in Z#####-His.sub.6 format yielded good production levels of soluble product and the purity of produced batches was estimated to exceed 95% by SDS-PAGE analysis. The LC/MS analysis verified the correct molecular weight for all pure Z variant molecules.
(65) Off-Rate Ranking by Biosensor Analysis:
(66) The ten Z variants expressed as described above were injected over a sensor chip surface immobilized with human HER3-Fc, and dissociation was followed during 30 min. All Z variant molecules generated similar binding curves to HER3 with greatly reduced off-rates compared to the precursor variant Z05417 (
(67) Biosensor Analysis of HER3 Binding Before and after Heat Treatment:
(68) The interaction between HER3 and the Z variants Z08698 and Z08699 before and after heat treatment at 90° C. was evaluated by surface plasmon resonance (SPR) technology. The Z variant molecules were injected, before and after heating, over immobilized human and mouse HER3-Fc, and the obtained binding curves were compared. As shown in
(69) Circular Dichroism Spectroscopy:
(70) From the VTM, the Tm for Z08698 and Z08699 were determined to be 64° C. and 65° C., respectively (
(71) Affinity Determination by Biosensor Analysis:
(72) The dissociation constants, K.sub.D, of the Z variants Z08698 and Z08699 were determined using biosensor technology. A dilution series of each binder was injected over immobilized human HER3-Fc and K.sub.D-values were determined by non-linear regression to a one-site binding model. The obtained affinities were estimated to 50 pM for Z08698 and 21 pM for Z08699 (Table 2), representing an affinity improvement of approximately 30 times when comparing the strongest binder Z08699 to the reference polypeptide Z05417. The reduced K.sub.D-values of the affinity matured Z variants are due to the significantly slower dissociation rates compared to Z05417, which in turn are ascribed to a successful incorporation of off-rate selections in the sorting procedure described in Example 2.
(73) TABLE-US-00002 TABLE 2 Affinities of two Z variants for human HER3 as determined by SPR K.sub.D k.sub.on k.sub.off Z variant (pM, mean ± SD).sup.a (M.sup.−1s.sup.−1, mean).sup.a (s.sup.−1, mean).sup.a Z08698 50 ± 5.0 8.3 × 10.sup.5 4.1 × 10.sup.−5 Z08699 21 ± 2.4 1.9 × 10.sup.6 3.9 × 10.sup.−5 .sup.aPerformed with duplicates of each concentration on the same day
Example 4
In Vitro Cell Assay Assessing the HER3 Specific Binding of Z Variants
(74) In this Example, the Z variants Z08698 and Z08699 with a C-terminal His.sub.6-tag were radiolabeled with .sup.99mTc and their in vitro cell binding properties to a range of different cancer cell lines were analyzed.
(75) Materials and Methods
(76) Labeling of Z Variants:
(77) The Z variants Z08698 and Z08699 with a C-terminal His.sub.6-tag, produced as described in Example 3, were labeled with .sup.99mTc at the His.sub.6-tag using an IsoLink kit (Covidien) as described previously for other Z variants (Orlova et al, Journal of Nuclear Medicine 47: 512-519 (2006)). In brief, 500 μl (200-320 MBq) of .sup.99mTc-pertechnetate solution eluted, with sterile 0.9% NaCl, from an Ultra-TechneKow generator (Covidien) was added to the IsoLink carbonyl labeling agent of the IsoLink kit. The mixture was incubated at 100° C. for 20 min. 40 μl of the mixture was transferred to solutions containing respective Z variant (50 μg, appr. 6.8 nmol, in 40 μl PBS) and incubated at 50° C. The labeling yield after incubation for 60 min was analyzed by instant thin-layer chromatography (ITLC; using Tec-Control Chromatography strips, DARK GREEN from Biodex Medical Systems, cat. no. 150-771) and elution with PBS. The distribution of radioactivity along the thin layer chromatography strips was measured on a CYCLONE Storage Phosphor System and analyzed using the OPTIQUANT image analysis software (PerkinElmer). The labeled Z molecules were purified using NAP-5 desalting columns (GE Healthcare), pre-equilibrated and eluted with PBS. The purity of each preparation was assessed using ITLC cross-validated by SDS-PAGE.
(78) Binding Specificity of Labeled Z Variants to HER3 Expressing Cells:
(79) The specificity of .sup.99mTc(CO).sub.3-Z08698-His.sub.6 and .sup.99mTc(CO).sub.3-Z08699-His.sub.6 for binding to HER3-expressing cells was evaluated using LS174T colorectal carcinoma, NCI-N87 gastric carcinoma, MCG7 breast carcinoma, LNCaP and DU-145 prostate cancer cell lines (American Type Tissue Culture Collection, ATCC, via LGC Promochem, Boras, Sweden) The in vitro specificity test was performed according to methods described previously (Orlova et al, (2006), supra). Briefly, a solution of radiolabeled Z variant molecules (at 1 nM) was added to 6 Petri dishes (each containing approximately 2×10.sup.6 cells). For blocking, 0.7 μM of non-labeled Z variant molecule was added 15 min before the radiolabeled conjugate to saturate the receptors. The cells were incubated for 1 h in a humidified incubator at 37° C. Thereafter, the media was collected and the cells were detached using trypsin-EDTA solution (0.25% trypsin, 0.02% EDTA in buffer, Biochrom AG, Berlin, Germany). The radioactivity in both media and cells was measured using an automated gamma-counter equipped with a 3-inch NaI(TI) detector (1480 WIZARD, Wallac Oy, Turku, Finland), and the fraction of cell-bound radioactivity was calculated. The data on cellular uptake was statistically assessed by an unpaired, two-tailed t-test using GraphPad Prism (version 4.00 for Windows GraphPad Software, San Diego Calif. USA) in order to determine any significant differences (p<0.05).
(80) Results
(81) Labeling of Z Variants:
(82) Radiolabeling using an IsoLink kit provided a yield of 43±6% for .sup.99mTc(CO).sub.3-Z08698-His.sub.6 and 73±12% .sup.99mTc(CO).sub.3-Z08699-His.sub.6. After purification with disposable NAP-5 columns, the purity was more than 97% for both labeled conjugates.
(83) Binding Specificity of Labeled Z Variants to HER3 Expressing Cells:
(84) Binding specificity tests were performed to assess if the binding of .sup.99mTc(CO).sub.3-Z08698-His.sub.6 and .sup.99mTc(CO).sub.3-Z08699-His.sub.6 to HER3-expressing cells was receptor mediated. Saturation of the receptors by pre-incubation with the same, but unlabeled, Z variant molecules, significantly (p<0.05) decreased the binding of the radiolabeled Z variant molecules, suggesting specific binding (
Example 5
In Vivo Biodistribution Studies of HER3 Binding Z Variants
(85) This Example describes in vivo studies performed in mice using radiolabeled conjugates of Z08698 and Z08699, demonstrated in Example 3 to cross-react with murine HER3. First, normal mice were used to study the biodistribution properties, as well as to test the specificity of in vivo accumulation of the two radiolabeled Z variant molecules in organs where HER3 is normally expressed, such as in lung, liver, stomach, small intestines and salivary gland. Second, the biodistribution and tumor targeting properties of radiolabeled Z08699 were further assessed in nude mice bearing a prostate cancer xenograft.
(86) Materials and Methods
(87) The biodistribution studies were performed using .sup.99mTc(CO).sub.3-Z08698-His.sub.6 and .sup.99mTc(CO).sub.3-Z08699-His.sub.6 Z variants labeled as described in Example 4. The animal studies were planned and performed in accordance with national legislation on laboratory animals' protection and approved by the local ethics committee for animal research.
(88) Biodistribution Studies in Normal NMRI Mice:
(89) .sup.99mTc(CO).sub.3-Z08698-His.sub.6 or .sup.99mTc(CO).sub.3-Z08699-His.sub.6 (65 kBq in 100 μl PBS per mouse) was intravenously injected in a group of four female NMRI mice (average weight 24.5±1.6 g). The injected protein dose was adjusted by dilution with non-labeled Z variant molecule to 1 μg (0.13 nmol) or 10 μg (1.3 nmol) per mouse. At 4 h post injection (pi), a group of four mice were sacrificed by injection of a lethal dose of anesthesia (20 μl of Ketalar-Rompun per gram body weight; Ketalar (50 mg/ml, Pfizer); Rompun (20 mg/ml, Bayer)) followed by heart puncture and exsanguination with a syringe rinsed with heparin (5000 IE/ml, Leo Pharma). Samples of blood, lung, liver, spleen, stomach, small intestines, kidney, uterus, salivary gland, muscle and bone were collected and weighed, and their radioactivity was measured using an automated gamma-counter equipped with a 3-inch NaI(TI) detector (1480 WIZARD, Wallac Oy, Turku, Finland). Technetium-99m radioactivity was measured in the energy range of 100-160 keV. The data were corrected for background. The tissue uptake was calculated as percent of injected radioactivity per gram (% IA/g). Radioactivity in carcass was calculated as % IA per whole sample. The biodistribution data was statistically assessed by an unpaired, two-tailed t-test using GraphPad Prism (version 4.00 for Windows, GraphPad Software) in order to determine any significant differences (p<0.05).
(90) Biodistribution Studies in Nude Tumor Bearing Mice:
(91) HER3-expressing prostate cancer xenografts were used to study the in vivo tumor targeting properties of .sup.99mTc(CO).sub.3-Z08699-His.sub.6. LNCaP cells (6×10.sup.6) were implanted in the right hind leg of male BALB/C nu/nu mice in 50% Matrigel. The biodistribution experiments were performed 4 weeks after implantation, at tumor weights of 0.8±0.4 g. The average animal weight was 20.1±0.6 g at the time of the experiment. To evaluate if the uptake in HER3 expressing organs is saturable, .sup.99mTc(CO).sub.3-Z08699-His.sub.6 (85 kBq in 100 μl PBS per mouse) was intravenously injected in a group of three mice. The injected protein dose was adjusted by dilution with non-labeled Z08699-His.sub.6 molecule to 0.1 μg (0.013 nmol) or 1 μg (0.13 nmol) per mouse. The animals were sacrificed 6 h after injection, and the biodistribution was measured and analyzed as described above.
(92) Results
(93) Biodistribution Studies in Normal Mice:
(94) The biodistribution of .sup.99mTc(CO).sub.3-Z08698-His.sub.6 and .sup.99mTc(CO).sub.3-Z08699-His.sub.6 was assessed 4 h after injection in female NMRI mice. Both conjugates were rapidly cleared from blood and non-HER3-expressing tissues, such as bone and muscle. In HER3-expressing tissues (lung, liver, stomach, small intestines and salivary gland), the uptake (% IA/g) of both conjugates was lower at the higher injected protein dose (10 μg, 1.3 nmol). This result indicates a saturable uptake, which is a strong evidence of HER3-specific targeting.
(95) Biodistribution Studies in Tumor Bearing Mice:
(96) The biodistribution data of .sup.99mTc(CO).sub.3-Z08699-His.sub.6 in nude mice bearing LNCaP xenografts are presented in
Example 6
Production of a Selection of HER3 Binding Z Variants in Fusion with an Albumin Binding Domain
(97) In this Example, two HER3 binding Z variants (Z08698 and Z08699) from the on-cell affinity ranking described in Example 2 and the two reference Z variants Z05416 and Z05417 were recloned as fusions with the albumin binding domain PP013 (SEQ ID NO:109), and purified from E. coli cell extracts.
(98) Materials and Methods
(99) Subcloning of Z Variants:
(100) This was performed essentially as described in WO2009/077175 for other Z variants described therein. Monomeric Z variants were amplified from pAY01449 vectors. A subcloning strategy for construction of Z variant molecules with N-terminal and/or C-terminal fusions was applied using standard molecular biology techniques. A PCR was performed using different primer pairs and the resulting gene fragments were purified and hybridized in ligase buffer. The hybridized gene fragments were subcloned in the pAY03362 vector, providing a C-terminal PP013 fusion. The HER3 binding Z variants were subcloned as monomers, and the exact constructs encoded by the expression vectors were MGSSLQ-[Z#####]-VDSS-[PP013] (SEQ ID NO: 123).
(101) Cultivation and Purification:
(102) E. coli BL21(DE3) cells (Novagen) were transformed with plasmids containing the monomeric gene fragment of each respective Z variant and cultivated at 37° C. in 1 I of TSB+YE medium (Tryptic Soy Broth with Yeast Extract) supplemented with 50 μg/ml kanamycin. At OD.sub.600=1, IPTG at a final concentration of 0.17 mM was added to induce protein expression and the cultivation was incubated at 37° C. for another 5 h. The cells were harvested by centrifugation. Cell pellets harboring the Z variants were resuspended in TST buffer (25 mM Tris-HCl, 1 mM EDTA, 200 mM NaCl, 0.05%, Tween 20, pH 8.0) with an addition of 7 U/ml BENZONASE (Merck, cat. no. 1.01654.001) and disrupted by ultrasonication. The lysates were clarified by centrifugation (>20 min, 25000 g, 4° C.) and loaded on 1 ml pre-packed affinity agarose, pre-equilibrated with TST buffer. After wash with 5 column volumes (CV) TST buffer, followed by 3 CV 5 mM NH.sub.4Ac pH 5.5, bound Z variants were eluted with 2 CV 0.1 M HAc. Each Z variant was transferred to PBS (2.68 mM KCl, 0.47 mM KH.sub.2PO.sub.4, 137 mM NaCl, 8.1 mM Na.sub.2HPO.sub.4, pH 7.4) on PD-10 Desalting Columns (GE Healthcare, cat. no. 17-0851-01). Protein concentrations were determined by measuring the absorbance at 280 nm, using a NANODROP ND-1000 spectrophotometer, and using the extinction coefficient of the respective protein. Purity of the final products was analyzed by SDS-PAGE stained with Coomassie Blue. Identity of each purified protein variant was determined using HPLC-MS analysis on an Agilent 1100 LC/MSD system (Agilent Technologies).
(103) Results
(104) Subcloning of Z Variants:
(105) The Z variants were chosen for subcloning in the expression vector pAY03362. The cloning resulted in four fusion proteins comprising one of the four monomers Z05416, Z05417, Z08698 and Z08699 in fusion with the albumin binding domain PP013.
(106) Cultivation and Purification:
(107) The two inventive Z variants Z08698 and Z08699, constructed as monomers and with a C-terminal ABD, expressed well in E. coli. The amount of affinity-purified Z variants from 2 g bacterial pellets, determined spectrophotometrically by measuring the absorbance at 280 nm, ranged from 3 mg to 6 mg for the different Z variants. Purity of the produced Z variants was estimated to exceed 95% as assessed by SDS-PAGE analysis. The correct molecular weight of each protein variant was confirmed by HPLC-MS.
Example 7
Evaluation of the Inhibitory Capacity of HER3 Binding Z Variants in an In Vitro Proliferation Assay
(108) In this Example, Z variants Z08698 and Z08699, fused at their C-termini to PP013 as described in Example 6, were tested for their ability to inhibit heregulin induced proliferation in an in vitro assay using MCF-7 cells.
(109) Materials and Methods
(110) The Z variants Z08698, Z08699, Z05416 and Z05417, all produced as described in Example 6 with a C-terminal albumin binding fusion partner, were tested. The cell line MCF-7 (ATCC HTB-22) was propagated as recommended in complete medium (RPMI 1640 medium with L-glut (Lonza) supplemented with sodium pyruvate (Lonza), non-essential amino acids (Lonza), penicillin/streptomycin (Lonza) and 10% fetal calf serum (FCS) (Gibco)). At the day of experiment, the cells were washed twice in RPMI 1640 without supplements and resuspended in assay medium (RPMI 1640 medium with L-glut containing sodium pyruvate, non-essential amino acids, penicillin/streptomycin, 9 pM recombinant human serum albumin (rHSA, Novozymes)+2% dialysed FCS (Gibco)). The ability of the Z variants to block heregulin (HRG) induced proliferation was analyzed by mixing the Z variants with 200 pM HRG (NRG1-J31/HRG1-J31 EGF domain, R&D Systems) in assay medium. The molecules were titrated in a 5-fold dilution series with a fixed concentration of HRG (200 pM). The titration was performed in 96-well cell culture plates in a volume of 100 μl. 1500 cells were added per well (100 μl) and plates were incubated at 37° C., 5% CO.sub.2 for five days. After incubation, determination of the number of living cells in each well was performed using cell counting kit-8 (CCK-8, Fluka, Sigma Aldrich). 19 μl of CCK-8 cell proliferation reagent diluted two times in RPMI 1640 medium was added per well and absorbance was measured after 4 h at 450 nm using a microplate reader (Victor3, Perkin Elmer). The data on cell growth was assessed by non-linear regression to a four-parameter dose-response curve, and the half maximal inhibitory concentration (IC50) was determined using GraphPad Prism (version 5.01 for Windows, GraphPad Software).
(111) Results
(112) Evaluation in HRG Induced Proliferation Assay:
(113) The results are shown in
Example 8
In Vitro Cell Assay Assessing the Inhibitory Capacity of HER3 Binding Z Variants in a HER3 Phosphorylation Assay
(114) In this Example, Z variants Z08698 and Z08699 produced with a C-terminal albumin binding domain were tested for their ability to inhibit HER3 phosphorylation.
(115) Materials and Methods
(116) The Z variants Z08698 and Z08699 were produced as described in Example 6 with a C-terminal PP013 fusion partner and tested. The cell line MCF-7 was propagated as recommended in complete medium.
(117) Cell Lysate Production:
(118) MCF-7 cells were seeded in 60×15 mm cell culture dishes (Corning) at a concentration of 1×10.sup.6 cells/5 ml and allowed to grow for 24 h in complete medium. The medium was exchanged for assay medium 4 h prior to the start of the experiment. Z variants Z08698 and Z08699 were diluted to 4, 40 and 400 nM in assay medium with a fixed concentration of HRG (4 nM) and incubated with the cells for 10 min at 37° C. The cells were kept on ice and washed twice with ice cold PBS. The cells were loosened with a cell scraper in 2 ml of ice cold PBS with 1 mM activated sodium orthovanadate (Sigma), transferred to a tube and centrifugated at 400 g for 3 min in a pre-cooled (4° C.) centrifuge. The supernatant was discarded and 100 μl lysis buffer (1% NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate) was added per 10.sup.6 cells. After a 30 min incubation at 4° C., the samples were centrifuged in eppendorf tubes at 13000×g for 15 min in a pre-cooled (4° C.) centrifuge. The supernatant from each tube was collected and used in phospho-HER3 ELISA as follows.
(119) Phospho-HER3 ELISA:
(120) Human Phospho-ErbB3 ELISA kit (R&D Systems) for detection of phosphorylated HER3 was used according to the manufacturer's instructions. 96-well half area plates were coated with an anti-HER3 antibody, 4 μg/ml in PBS at room temperature overnight. The plate was washed and blocked with 1% BSA in PBS for 2 h at room temperature. After washing, 50 μl of cell lysate diluted 1:20 in “diluent #12” (1% NP-40, 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 2 mM EDTA, 1 mM activated sodium orthovanadate) was added to each well. The plate was incubated for 2 h, washed and incubated with HRP-labeled anti phospho-tyrosine antibody, diluted 1:2000 in “diluent #14” (20 mM Tris, 137 mM NaCl, 0.05% Tween 20, 0.1% BSA, pH 7.2-7.4), for 2 h. The plate was washed and substrate added (R&D Systems). After about 20 min, the reaction was stopped with 2 M H.sub.2SO.sub.4 and the plate was read using a microplate reader (Victor3, Perkin Elmer) at 450 and 570 nm.
(121) Results
(122) HER3 Phosphorylation Assay:
(123) The results are shown in
Example 9
Evaluation of the Inhibitory Capacity of Dimeric HER3 Binding Z Variants
(124) In this Example, dimeric Z variants comprising Z08698 in fusion with the albumin binding PP013 in the format Z-PP013-Z was tested for their ability to inhibit heregulin induced signaling in an in vitro assay using MCF-7 cells. Two different linker lengths with one or four repeats, respectively, of GGGGS between the Z and the albumin binding moieties were assessed i.e. Z08698-G.sub.4S-PP013-G.sub.4S-Z08698 (SEQ ID NO: 126) and Z08698-(G.sub.4S).sub.4-PP013-(G.sub.4S).sub.4-Z08698 (SEQ ID NO: 127).
(125) Materials and Methods
(126) Cloning and Production:
(127) Subcloning of dimeric Z variants was performed essentially as described in Example 6 and applying standard molecular biology techniques using primers incorporating NdeI and AscI restriction sites. The HER3 binding Z variants were subcloned as dimers in fusion with PP013, and the exact constructs encoded by the expression vectors were M-[Z08698]-GAP(GGGGS)TS-[PP013]-GT(GGGGS)PR-[Z08698] (SEQ ID NO: 124) and M-[Z08698]-GAP(GGGGS).sub.4TS-[PP013]-GT(GGGGS).sub.4PR-[Z08698] (SEQ ID NO: 125), respectively. The protein variants were expressed in E. coli and purified essentially as described in Example 6, but with the addition of a preparative reverse phase high-performance liquid chromatography (RP-HPLC) purification step after the affinity chromatography step and prior to final buffer exchange to PBS.
(128) In Vitro Cell Assay:
(129) MCF-7 cells (ATCC HTB-22) were trypsinated and seeded at 25000 cells per well in an EnSpire-LFC microplate (Perkin Elmer, cat no 6055408) and allowed to grow for 20 h at 37° C. in complete medium. At the day of experiment, the cells were washed once with Hank's balanced salt solution (HBSS; Sigma, cat. no. H9269). 100 μl HBSS was added per well and the plates were incubated at ambient temperature for 1.5 h inside the EnSpire Instrument (Perkin Elmer) to be used for assay readout. The ability of Z08698-G.sub.4S-PP013-G.sub.4S-Z08698 (SEQ ID NO: 126) and Z08698-(G.sub.4S).sub.4-PP013-(G.sub.4S).sub.4-Z08698 (SEQ ID NO: 127), respectively, to block heregulin induced signaling was analyzed by mixing these Z-PP013-Z variants with 1 nM HRG in HBSS. The monomeric Z08698-PP013 was included for comparison. The molecules were titrated in a 5-fold dilution series with a fixed concentration of HRG (1 nM) in a final assay volume of 130 μl/well. The redistribution of dynamic mass upon addition of stimuli was recorded by an EnSpire Instrument every 60 s for 1 h. The half maximal inhibitory concentrations (IC50) were determined from the dose response curves.
(130) Results
(131) The obtained IC50 values for Z08698-G.sub.4S-PP013-G.sub.4S-Z08698 (SEQ ID NO: 126), Z08698-(G.sub.4S).sub.4-PP013-(G.sub.4S).sub.4-Z08698 (SEQ ID NO: 127) and Z08698-PP013 were 0.7 nM, 0.6 nM and 1 nM respectively. Thus, the capacity to inhibit heregulin induced signaling was increased by the dimeric constructs in the format Z-PP013-Z. The effect of linker length between the Z and albumin binding moieties was marginal. The IC50 value for the Z08698-PP013 variant (1 nM) is in line with the result obtained in the proliferation assay described in Example 7, where it was shown to be superior to the reference polypeptide Z05417-PP013.
Example 10
Imaging of HER3-Expressing Xenografts in Mice Using 99mTc(CO)3-Labeled Z Variant
(132) In this Example, the feasibility of using a radiolabelled HER3 specific Z variant for imaging was investigated. Z08699 with a HEHEHE-tag on the N-terminus was labeled with .sup.99mTc(CO).sub.3 and injected into LS174T colorectal carcinoma xenograft mouse. Tumors were visualized by microSPECT/CT 4 h post injection.
(133) Materials and Methods
(134) Cloning, Production and Labeling of Z Variant:
(135) DNA encoding Z08699 was amplified by PCR using primers incorporating NdeI and XhoI restriction sites and codons for an N-terminal HEHEHE-tag. Subcloning and production of HEHEHE-Z08699 (SEQ ID NO: 128) was performed essentially as described in Example 3, but with the addition of a preparative reverse phase high-performance liquid chromatography (RP-HPLC) purification step after the IMAC purification. A histidyl-glytamyl-histidyl-glytamyl-histidyl-glytamyl-(HEHEHE)-tag was selected instead of a His.sub.6 tag because it has a more favorable biodistribution profile, in particular reduced hepatic uptake, but yet allows IMAC-purification (Tolmachev et al, Bioconjug Chem 21:2013-2022 (2010)). Labeling with .sup.99mTc(CO).sub.3 using an IsoLink kit was performed as described in Example 4.
(136) In Vivo Imaging:
(137) A Balb/c nu/nu mouse bearing a subcutaneous LS174T colorectal carcinoma xenograft, was injected with .sup.99mTc(CO).sub.3-HEHEHE-Z08699 (1.6 MBq/1 μg). 4 h post injection, the animal was euthanized and the urinary bladder was excised post-mortem to improve image quality. Static whole body tomographical examinations was then performed by microSPECT (FOV: 8 cm, 75A10 collimators, acquisition over 200-250 keV, 32 projections). The animal was examined by CT for anatomical correlation.
(138) Results
(139) A microSPECT image acquired 4 h after administration of .sup.99mTc(CO).sub.3-HEHEHE-Z08699 to tumor bearing mouse (LS174T colorectal carcinoma xenograft) is presented in
Example 11
Imaging of HER3-Expressing Xenografts in Mice Using 111In-Labeled Z Variants
(140) In this Example, the feasibility of using radiolabeled HER3 specific Z variants for imaging was investigated further. The NOTA chelator, forming a stable complex with a number of radionuclides suitable for SPECT or PET imaging, was conjugated to HEHEHE-Z08698 (SEQ ID NO: 129) and HEHEHE-Z08699 (SEQ ID NO: 128) via a unique C-terminal cysteine. The molecules were labeled with .sup.111In and injected into BT474 breast carcinoma xenograft mice. Tumors were visualized by SPECT gamma camera imaging 4 h post injection.
(141) Materials and Methods
(142) Cloning and Production of NOTA Coupled Z Variants:
(143) HEHEHE-Z08698 (SEQ ID NO: 129) and HEHEHE-Z08699 (SEQ ID NO: 128), each with a C-terminal cysteine residue were cloned and expressed essentially as described in Example 3. Harvested cells were resuspended in 1×PBS and disrupted by the use of a French press. The samples were heat treated up to 70° C. and incubated for 10 min, followed by cooling on ice for 10 min. The lysates were clarified by centrifugation (10 min, 30000 g, 4° C.). The cysteines of the Z variants were reduced with DTT, 20 mM for 30 min at 40° C. Excess DTT was removed by buffer exchange on PD-10 columns to 20 mM NH.sub.4 acetate, pH 5.5. The NOTA conjugation was performed with a three-fold molar excess of chelator, maleimide-NOTA (CheMatech, cat. no. C101). The mixture was incubated for 40 min at 40° C. Purification from non-conjugated chelators was performed by RP-HPLC. The correct molecular weight of each NOTA-coupled Z variant was confirmed by HPLC-MS and CD measurements were performed to verify preserved structural integrity.
(144) Labelling with .sup.111In:
(145) HEHEHE-Z08698-NOTA (SEQ ID NO: 130) or HEHEHE-Z08699-NOTA (SEQ ID NO: 131) (40 μg, 6 nmol) in 100 μl 20 mM NH.sub.4 acetate, pH 5.5 was mixed with 54 μl .sup.111In-chloride solution (40 MBq). The mixture was incubated at 85° C. for 1 h. The labeling efficiency was analyzed by ITLC (as described in Example 4) eluted with 0.2 M citric acid, pH 2.0. The conjugates were purified using disposable NAP-5 columns (GE Healthcare) according to manufacturer's instructions.
(146) In Vivo Imaging:
(147) Tumor-bearing mice (BT474 breast carcinoma xenograft) were injected with 1 μg (0.8 MBq) .sup.111In-HEHEHE-Z08698-NOTA (SEQ ID NO: 132) or .sup.111In-HEHEHE-Z08699-NOTA (SEQ ID NO: 133). 4 h pi, the animals were euthanized and the urinary bladders were excised post-mortem to improve image quality. Static planar imaging was performed using a GE Infinia gamma camera equipped with a medium energy general purpose (MEGP) collimator. Static image (20 min) was obtained with a zoom factor of 3 in a 256×256 matrix.
(148) Results
(149) The correct molecular weight of each NOTA-conjugated Z variant was confirmed by HPLC-MS and purity was found to exceed 95%. CD measurements verified preserved helical structure as well as reversible folding after heating up to 90° C. The radiochemical yields of .sup.111In-HEHEHE-Z08698-NOTA (SEQ ID NO: 132) and .sup.111In-HEHEHE-Z08699-NOTA (SEQ ID NO: 133) were 97% and 96%, respectively.
(150) Gamma-camera images of tumor-bearing mice 4 h post injection of .sup.111In-HEHEHE-Z08698-NOTA (SEQ ID NO: 132) and .sup.111In-HEHEHE-Z08699-NOTA (SEQ ID NO: 133), respectively, are shown in
(151) To conclude, the results disclosed in Examples 10 and 11 show that radionuclide imaging of HER3 expression in malignant tumors is feasible using the HER3 specific high affinity Z variants disclosed herein, despite low HER3 expression in tumors and background expression in normal tissues. Improved imaging contrast may be obtained by optimizing labeling chemistry and tracer dosing.
Example 12
Inhibition of Tumor Growth In Vivo by Administration of HER3 Binding Z Variant
(152) To show that a HER3 binding Z variant as disclosed herein inhibits tumor growth in vivo, the Z variant is administered to xenografted mice and tumor growth is monitored. One useful such HER 3-expressing tumor model is the ACHN xenograft model.
(153) To obtain cells for xenograft experiments, ACHN cells (CRL-1611, LGC standards) are cultured in vitro in MEM medium (Lonza, cat no 12-611) containing 10% fetal calf serum. ACHN tumors are established by subcutaneous injection of 5-10×10.sup.6 ACHN cells into the right flank of NMRI nu/nu mice (Charles River). Tumor volume is measured using calipers to measure the length and width of tumors three times a week. The tumor volume is calculated as length×width.sup.2×0.44. According to generally accepted principles, the study is started when tumor volume reaches approximately 200 mm.sup.3. Mice are randomized into groups containing similar size distribution of the tumors.
(154) To inhibit tumor growth with a HER3 binding Z variant, mice (10 per group) are injected intravenously with 20, 2 or 0.2 mg/kg of endotoxin free Z variant or vehicle control (PBS buffer). The injections are repeated three times per week during three weeks. The therapeutic effect is followed by measuring tumor volume three times weekly using calipers, and the body weight of each mouse is simultaneously recorded. The difference in average tumor volume between treated and vehicle groups at the end of study is assessed by Students t-test.
(155) The results of the experiment above is expected to show a dose dependent inhibition of tumor growth by the HER3 binding Z variant disclosed herein.
ITEMIZED LISTING OF EMBODIMENTS
(156) 1. HER3 binding polypeptide, comprising a HER3 binding motif (BM), which motif consists of an amino acid sequence selected from: i) EKYX.sub.4AYX.sub.7EIW X.sub.11LPNLTX.sub.17X.sub.18QX.sub.20 AAFIGX.sub.26LX.sub.28D (SEQ ID NO: 110)
wherein, independently of each other,
X.sub.4 is selected from A, E, L, M, N Q, R, S and T;
X.sub.7 is selected from F and Y;
X.sub.11 is selected from E and Q;
X.sub.17 is selected from K, N, R and V;
X.sub.18 is selected from F, M, N, R, T, Y and W;
X.sub.20 is selected from A and K;
X.sub.26 is selected from K and S;
X.sub.28 is selected from E and Q; and ii) an amino acid sequence which has at least 96% identity to the sequence defined in i).
(157) 2. HER3 binding polypeptide according to item 1, wherein in sequence i), independently from each other,
(158) X.sub.4 is selected from A, E, M, N, Q, S and T;
(159) X.sub.7 is selected from F and Y;
(160) X.sub.11 is Q;
(161) X.sub.17 is selected from K and R;
(162) X.sub.18 is selected from M, Y and W;
(163) X.sub.20 is K;
(164) X.sub.26 is K;
(165) X.sub.28 is Q.
(166) 3. HER3 binding polypeptide according to item 1, wherein X.sub.4 in sequence i) is selected from A, E, M, N, Q, S and T.
(167) 4. HER3 binding polypeptide according to any preceding item, wherein X.sub.4 in sequence i) is selected from N and Q.
(168) 5. HER3 binding polypeptide according to item 4, wherein X.sub.4 in sequence i) is N.
(169) 6. HER3 binding polypeptide according to item 4, wherein X.sub.4 in sequence i) is Q.
(170) 7. HER3 binding polypeptide according to any preceding item, wherein X.sub.11 in sequence i) is Q.
(171) 8. HER3 binding polypeptide according to any preceding item, wherein X.sub.17 in sequence i) is selected from K, N and R, such as selected from K and R.
(172) 9. HER3 binding polypeptide according to item 8, wherein X.sub.17 in sequence i) is K.
(173) 10. HER3 binding polypeptide according to item 8, wherein X.sub.17 in sequence i) is R.
(174) 11. HER3 binding polypeptide according to any preceding item, wherein X.sub.18 in sequence i) is selected from M, Y and W.
(175) 12. HER3 binding polypeptide according to any item 11, wherein X.sub.18 in sequence i) is selected from Y and W.
(176) 13. HER3 binding polypeptide according to item 12, wherein X.sub.18 in sequence i) is Y.
(177) 14. HER3 binding polypeptide according to item 12, wherein X.sub.18 in sequence i) is W.
(178) 15. HER3 binding polypeptide according to item 11, wherein X.sub.18 in sequence i) is M.
(179) 16. HER3 binding polypeptide according to any preceding item, wherein X.sub.17X.sub.18 in sequence i) is selected from KW, KY, KM and RY.
(180) 17. HER3 binding polypeptide according to any preceding item, wherein X.sub.20 in sequence i) is K.
(181) 18. HER3 binding polypeptide according to any preceding item, wherein X.sub.26 in sequence i) is K.
(182) 19. HER3 binding polypeptide according to any preceding item, wherein X.sub.28 in sequence i) is Q.
(183) 20. HER3 binding polypeptide according to any preceding item, wherein sequence i) fulfills at least two of the following four conditions I, II, III and IV:
(184) I) X.sub.ii is Q;
(185) II) X.sub.17X.sub.18 is selected from KW, KY, KM and RY;
(186) III) X.sub.20 is K;
(187) IV) X.sub.28 is Q.
(188) 21. HER3 binding polypeptide according to item 20, which fulfills at least three of said four conditions I, II, III and IV.
(189) 22. HER3 binding polypeptide according to item 21, which fulfills all of said four conditions I, II, III and IV.
(190) 23. HER3 binding polypeptide according to any preceding item, wherein sequence i) is selected from any one of SEQ ID NO:1-35.
(191) 24. HER3 binding polypeptide according to item 23, wherein sequence i) is selected from any one of SEQ ID NO:1-10.
(192) 25. HER3 binding polypeptide according to item 24, wherein sequence i) is selected from SEQ ID NO:1 and SEQ ID NO:2.
(193) 26. HER3 binding polypeptide according to item 25, wherein sequence i) is SEQ ID NO:2.
(194) 27. HER3 binding polypeptide according to item 25, wherein sequence i) is SEQ ID NO:1.
(195) 28. HER3 binding polypeptide according to any preceding item, wherein said HER3 binding motif forms part of a three-helix bundle protein domain.
(196) 29. HER3 binding polypeptide according to item 28, wherein said HER3 binding motif essentially forms part of two helices with an interconnecting loop, within said three-helix bundle protein domain.
(197) 30. HER3 binding polypeptide according to item 29, wherein said three-helix bundle protein domain is selected from bacterial receptor domains.
(198) 31. HER3 binding polypeptide according to item 30, wherein said three-helix bundle protein domain is selected from domains of protein A from Staphylococcus aureus or derivatives thereof.
(199) 32. HER3 binding polypeptide according to any preceding item, which comprises an amino acid sequence selected from: iii) K-[BM]-DPSQS X.sub.aX.sub.bLLX.sub.c EAKKL NDX.sub.dQ (SEQ ID NO: 111);
wherein
[BM] is a HER3 binding motif as defined by any one of items 1-27;
X.sub.a is selected from A and S;
X.sub.b is selected from N and E;
X.sub.c is selected from A, S and C;
X.sub.d is selected from A and S; and iv) an amino acid sequence which has at least 89% identity to the sequence defined in iii).
(200) 33. HER3 binding polypeptide according to item 32, wherein X.sub.a in sequence iii) is A.
(201) 34. HER3 binding polypeptide according to item 32, wherein X.sub.a in sequence iii) is S.
(202) 35. HER3 binding polypeptide according to any one of items 32-34, wherein X.sub.b in sequence iii) is N.
(203) 36. HER3 binding polypeptide according to any one of items 32-34, wherein X.sub.b in sequence iii) is E.
(204) 37. HER3 binding polypeptide according to any one of items 32-36, wherein X.sub.c in sequence iii) is A.
(205) 38. HER3 binding polypeptide according to any one of items 32-36, wherein X.sub.c in sequence iii) is S.
(206) 39. HER3 binding polypeptide according to any one of items 32-36, wherein X.sub.c in sequence iii) is C.
(207) 40. HER3 binding polypeptide according to any one of items 32-39, wherein X.sub.d in sequence iii) is A.
(208) 41. HER3 binding polypeptide according to any one of items 32-39, wherein X.sub.d in sequence iii) is S.
(209) 42. HER3 binding polypeptide according to item 32, wherein, in sequence iii), X.sub.a is A; X.sub.b is N; X.sub.c is A and X.sub.d is A.
(210) 43. HER3 binding polypeptide according to item 32, wherein, in sequence iii), X.sub.a is A; X.sub.b is N; X.sub.c is C and X.sub.d is A.
(211) 44. HER3 binding polypeptide according to item 32, wherein, in sequence iii), X.sub.a is S; X.sub.b is E; X.sub.c is S and X.sub.d is S.
(212) 45. HER3 binding polypeptide according to item 32, wherein, in sequence iii), X.sub.a is S; X.sub.b is E; X.sub.c is C and X.sub.d is S.
(213) 46. HER3 binding polypeptide according to any one of items 32-45, wherein sequence iii) is selected from any one of SEQ ID NO:36-70.
(214) 47. HER3 binding polypeptide according to item 46, wherein sequence iii) is selected from any one of SEQ ID NO:36-45.
(215) 48. HER3 binding polypeptide according to item 47, wherein sequence iii) is selected from SEQ ID NO:36 and SEQ ID NO:37.
(216) 49. HER3 binding polypeptide according to item 48, wherein sequence iii) is SEQ ID NO:37.
(217) 50. HER3 binding polypeptide according to item 48, wherein sequence iii) is SEQ ID NO:36.
(218) 51. HER3 binding polypeptide according to any one of items 1-32, which comprises an amino acid sequence selected from: v) YAK-[BM]-DPSQS SELLX.sub.c EAKKL NDSQA P (SEQ ID NO: 112);
wherein [BM] is a HER3 binding motif as defined in any one of items 1-27 and X.sub.c is selected from S and C; and vi) an amino acid sequence which has at least 90% identity to the sequence defined in v).
(219) 52. HER3 binding polypeptide according to any one of items 1-32, which comprises an amino acid sequence selected from: vii) FNK-[BM]-DPSQS ANLLX.sub.c EAKKL NDAQA P (SEQ ID NO: 113);
wherein [BM] is a HER3 binding motif as defined in any one of items 1-27 and X.sub.c is selected from A and C; and viii) an amino acid sequence which has at least 90% identity to the sequence defined in vii).
(220) 53. HER3 binding polypeptide according to any one of items 1-31, which comprises an amino acid sequence selected from: ADNNFNK-[BM]-DPSQSANLLSEAKKLNESQAPK (SEQ ID NO: 114); ADNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 115); ADNKFNK-[BM]-DPSVSKEILAEAKKLNDAQAPK (SEQ ID NO: 116); ADAQQNNFNK-[BM]-DPSQSTNVLGEAKKLNESQAPK (SEQ ID NO: 117); AQHDE-[BM]-DPSQSANVLGEAQKLNDSQAPK (SEQ ID NO: 118); VDNKFNK-[BM]-DPSQSANLLAEAKKLNDAQAPK (SEQ ID NO: 119); VDAKYAK-[BM]-DPSQSSELLAEAKKLNDAQAPK (SEQ ID NO: 120); and AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK (SEQ ID NO: 121); wherein [BM] is a HER3 binding motif as defined in any one of items 1-27.
(221) 54. HER3 binding polypeptide according to any one of items 1-51, which comprises an amino acid sequence selected from: ix) AEAKYAK-[BM]-DPSESSELLSEAKKLNKSQAPK (SEQ ID NO: 121);
wherein [BM] is a HER3 binding motif as defined in any one of items 1-27, and x) an amino acid sequence which has at least 91% identity to the sequence defined in ix).
(222) 55. HER3 binding polypeptide according to item 54, wherein sequence ix) is selected from SEQ ID NO:71-105.
(223) 56. HER3 binding polypeptide according to item 55, wherein sequence ix) is selected from SEQ ID NO:71-80.
(224) 57. HER3 binding polypeptide according to item 56, wherein sequence ix) is selected from SEQ ID NO:71 and SEQ ID NO:72.
(225) 58. HER3 binding polypeptide according to item 57, wherein sequence ix) is SEQ ID NO:72.
(226) 59. HER3 binding polypeptide according to item 57, wherein sequence ix) is SEQ ID NO:71.
(227) 60. HER3 binding polypeptide according to any preceding item, wherein the off-rate (k.sub.off) of the interaction between said HER3 binding polypeptide and human HER3 is at least four-fold reduced, when compared to the off-rate (k.sub.off) of the interaction between a comparative HER3 binding polypeptide comprising the amino acid sequence SEQ ID NO:107 and human HER3, as measured using the same experimental conditions.
(228) 61. HER3 binding polypeptide according item 60, wherein said off-rate (k.sub.off) is at least 8-fold reduced, such as at least 12-fold reduced, such as at least 15-fold reduced.
(229) 62. HER3 binding polypeptide according to item 61, wherein said off-rate (k.sub.off) is at least 20-fold reduced.
(230) 63. HER3 binding polypeptide according to any preceding item, wherein the K.sub.D value of the interaction between said HER3 binding polypeptide and human HER3 is at most 1×10.sup.−9 M, such as at most 1×10.sup.−10 M, such as at most 1×10.sup.−11 M.
(231) 64. HER3 binding polypeptide according to any preceding item, further comprising at least one additional amino acid residue.
(232) 65. Fusion protein or conjugate comprising a) a first moiety consisting of a HER3 binding polypeptide according to any preceding item; and b) a second moiety consisting of a polypeptide having a desired biological activity.
(233) 66. Fusion protein or conjugate according to item 65, wherein said desired biological activity is a therapeutic activity.
(234) 67. Fusion protein or conjugate according to item 65, wherein said desired biological activity is a binding activity.
(235) 68. Fusion protein or conjugate according to item 65, wherein said desired biological activity is an enzymatic activity.
(236) 69. Fusion protein or conjugate according to item 66, wherein the second moiety having a desired biological activity is a therapeutically active polypeptide.
(237) 70. Fusion protein or conjugate according to item 65, wherein the second moiety having a desired biological activity is selected from the group consisting of human endogenous enzymes, hormones, growth factors, chemokines, cytokines and lymphokines.
(238) 71. Fusion protein or conjugate according to item 67, wherein the second moiety having a binding activity is a binding polypeptide capable of selective interaction with a target molecule.
(239) 72. Fusion protein or conjugate according to item 71, wherein said target molecule is selected from the group consisting of albumin, HER3, HER2, EGFR, IGF1R, cMet, VEGFR and PDGFR.
(240) 73. Fusion protein or conjugate according to any one of items 65-72, comprising a further moiety consisting of a polypeptide having a further, desired biological activity, which may be the same as or different from that of the second moiety.
(241) 74. Fusion protein or conjugate according to item 73, wherein the second moiety is as defined in any one of items 66-70, and the further moiety is as defined in any one of items 71-72.
(242) 75. Fusion protein or conjugate according to item 73, wherein the second moiety and the further moiety each individually is as defined in any one of items 71-72.
(243) 76. HER3 binding polypeptide, fusion protein or conjugate according to any preceding item, further comprising a cytotoxic agent.
(244) 77. HER3 binding polypeptide, fusion protein or conjugate according to item 76, wherein the 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, taxol, ricin, bouganin, gelonin, pseudomonas exotoxin 38 (PE38), diphtheria toxin (DT), and their analogues, and derivates thereof and combinations thereof.
(245) 78. HER3 binding polypeptide, fusion protein or conjugate according to any preceding item further comprising a label.
(246) 79. HER3 binding polypeptide, fusion protein or conjugate according to item 78, wherein said label is selected from the group consisting of fluorescent dyes and metals, chromophoric dyes, chemiluminescent compounds and bioluminescent proteins, enzymes, radionuclides and particles.
(247) 80. 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.
(248) 81. HER3 binding polypeptide, fusion protein or conjugate according to item 80, wherein the polyaminopolycarboxylate chelator is 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid or a derivative thereof.
(249) 82. HER3 binding polypeptide, fusion protein or conjugate according to item 81, 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.
(250) 83. HER3 binding polypeptide, fusion protein or conjugate according to item 80, wherein the polyaminopolycarboxylate chelator is 1,4,7-triazacyclononane-1,4,7-triacetic acid or a derivative thereof.
(251) 84. HER3 binding polypeptide, fusion protein or conjugate according to item 80, wherein the polyaminopolycarboxylate chelator is diethylenetriaminepentaacetic acid or derivatives thereof.
(252) 85. HER3 binding polypeptide, fusion protein or conjugate according to any one of items 78-84, comprising a radionuclide suitable for medical imaging, said radionuclide being selected from the group consisting of .sup.99mTc, .sup.61Cu, .sup.64Cu, .sup.66Ga, .sup.67Ga, .sup.68Ga, .sup.110mIn, .sup.111In, .sup.44Sc and .sup.86Y, or with a radionuclide suitable for therapy, said radionuclide being selected from the group consisting of .sup.225Ac, .sup.212Bi, .sup.213Bi, .sup.67Cu, .sup.166Ho, .sup.177Lu, .sup.212Pb, .sup.149Pm, .sup.153Sm, .sup.227Th and .sup.90Y, wherein the radionuclide is complexed with the HER3 binding polypeptide via the chelating environment.
(253) 86. HER3 binding polypeptide, fusion protein or conjugate according to item 85, wherein the radionuclide is selected from the group consisting of .sup.99mTc, .sup.111In, .sup.64Cu and .sup.68Ga.
(254) 87. HER3 binding polypeptide, fusion protein or conjugate according to item 86, wherein the radionuclide is selected from .sup.99mTc and .sup.111In.
(255) 88. Composition comprising a HER3 binding polypeptide, fusion protein or conjugate according to any preceding item and at least one pharmaceutically acceptable excipient or carrier.
(256) 89. Composition according to item 88, further comprising at least one additional active agent.
(257) 90. Composition according to item 89, wherein said at least one additional active agent is a therapeutic agent.
(258) 91. Composition according to item 90, wherein said therapeutic agent is selected from the group consisting of immunostimulatory agents, radionuclides, toxic agents, enzymes, factors recruiting effector cells and photosensitizers.
(259) 92. HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1-87 or a composition according to any one of items 88-91 for use as a medicament, a diagnostic agent or a prognostic agent.
(260) 93. HER3 binding polypeptide, fusion protein, conjugate or composition for use according to item 92, wherein said polypeptide, fusion protein, conjugate or composition modulates HER3 signaling, such as inhibits HER3 signaling.
(261) 94. HER3 binding polypeptide, fusion protein, conjugate or composition for use according to item 92 or 93 in the treatment, diagnosis or prognosis of a HER3 related condition.
(262) 95. HER3 binding polypeptide, fusion protein, conjugate or composition for use according to item 94, wherein said HER3 related condition is cancer.
(263) 96. HER3 binding polypeptide, fusion protein, conjugate or composition for use according to item 95, wherein said cancer is selected from the group consisting of breast cancer, ovarian cancer and prostate cancer.
(264) 97. Method of detecting HER3, comprising providing a sample suspected to contain HER3, contacting said sample with a HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1-87 or a composition according to any one of items 88-91, and detecting the binding of the HER3 binding polypeptide, fusion protein, conjugate or composition to indicate the presence of HER3 in the sample.
(265) 98. Method for determining the presence of HER3 in a subject, the method comprising the steps: contacting the subject, or a sample isolated from the subject, with a HER3 binding polypeptide, fusion protein or conjugate according to any one of items 1-87 or a composition according to any one of items 89-91, and obtaining a value corresponding to the amount of the HER3 binding polypeptide, fusion protein, conjugate or composition that has bound in said subject or to said sample.
(266) 99. Method according to item 98, further comprising a step of comparing said value to a reference.
(267) 100. Method according to any one of items 97-99, wherein the method is performed in vivo.
(268) 101. Method according to any one of items 97-99, wherein the method is performed in vitro.
(269) 102. Method of in vivo imaging of the body of a subject having or suspected of having a cancer characterized by over expression of HER3, the method comprising the steps: administering a radiolabeled polypeptide, fusion polypeptide or conjugate according to any one of items 85-87, wherein the radionuclide is suitable for imaging, into the body of the mammalian subject; and obtaining one or more images, within 1-72 hours of administration of the radiolabeled polypeptide, of at least a part of the subject's body using a medical imaging instrument, said image(s) indicating the presence of the radionuclide inside the body.
(270) 103. Method according to item 98-102, wherein said subject is a mammalian subject, such as a human subject.
(271) 104. 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 1-87 or a composition according to any one of items 89-91.
(272) 105. Method according to item 104, wherein said HER3 binding polypeptide, fusion protein or conjugate or composition inhibits HER3 signaling.
(273) 106. Method according to item 104 or 105, wherein said HER3 related condition is cancer.
(274) 107. Method according to item 106, wherein said cancer is selected from the group consisting of breast cancer, ovarian cancer and prostate cancer.
(275) 108. Polynucleotide encoding an HER3 binding polypeptide or a fusion protein according to any one of items 1-75.
(276) 109. Expression vector comprising a polynucleotide according to item 108.
(277) 110. Host cell comprising an expression vector according to item 109.
(278) 111. Method of producing a polypeptide according to any one of items 1-75, comprising expressing a polynucleotide according to item 108.
(279) 112. Method of producing a polypeptide according to any one of items 1-75, comprising culturing a host cell according to item 110 under conditions permitting expression of said polypeptide from said expression vector, and isolating the polypeptide.