Anti-her2 polypeptide derivatives as new diagnostic molecular probes

Abstract

The present invention provides novel polypeptide derivatives that bind to human epidermal growth factor receptor 2 (HER2) and conjugates thereof, and their use as diagnostic agents, in particular for the early detection, patient stratification and treatment monitoring of cancer forms characterized by overexpression of HER2.

Description

Technical Field

[0001] This invention relates to novel polypeptide derivatives and conjugates that bind to human epidermal growth factor receptor 2 (HER2), and their use as diagnostic agents, particularly for the early detection, patient stratification, and treatment monitoring of cancer forms characterized by HER2 overexpression. Background Technology

[0002] In recent years, the discovery of biomarkers has become a dynamic and powerful approach due to their potential to identify the earliest events in pathological states in the fields of diagnosis and treatment. Developing probes capable of identifying targets in molecular pathways involved in disease states has also become extremely challenging. These probes must be able to improve diagnostic procedures to better apply current treatment regimens, improve treatment outcomes, and reduce overtreatment of patients.

[0003] The proto-oncogene HER2 or HER2 / neu (human epidermal growth factor receptor 2) encodes the cell surface receptor known as the HER2 protein or receptor. It is a member of the ErbB family of tyrosine kinase receptors, involved in the transmission of signals controlling normal cell growth and differentiation. Due to errors in the DNA replication system in tumor cells, the HER2 gene can be amplified, leading to overexpression of the HER2 protein on the cell surface. In particular, overexpression of the HER2 receptor occurs in 25-30% of primary human breast cancers. 1 It has been described as being associated with many other HER2-positive tumors, including, for example, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, and gastrointestinal tumors.

[0004] For these reasons, HER2 represents an important molecular target for many cancer treatments and diagnostic approaches. Among the methods for inhibiting the HER2 protein on the surface of tumor cells, several therapies using humanized variants of monoclonal antibodies (such as trastuzumab and pertuzumab) have been marketed in recent years for the treatment of HER2-positive tumors; however, despite clinical success, their use has been associated with some toxic effects and adverse side effects on healthy tissues, which is a cause for concern.

[0005] Generally, many antibodies are characterized by slow and inefficient tumor penetration, long biodistribution times, and slow clearance from the bloodstream. Furthermore, for diagnostic applications, long-lived radioactive isotopes are required for imaging at later time points, exposing patients to a high radiation burden. In addition, monoclonal antibodies may be immunogenic, thus ruling out repeated administration of routine diagnostic procedures.

[0006] Therefore, there remains a great need to continue to provide new molecules that can interact with the HER2 form.

[0007] These problems can be circumvented, for example, by using small molecules.

[0008] Recently, small binding peptides (4 to 20 kDa), such as affibody, fibronectin, and DARPin, have been described and have attracted interest as potential alternatives to antibodies for non-invasive imaging methods. Typically, these peptides have been shown to be biocompatible and low-immunogenic in vivo, highly selective, and capable of binding to and recognizing specific targets, exhibiting affinity constants (Ka) in the nanomolar range. d (), lower than antibodies, which typically have K values ​​in the nanomolar range. d Furthermore, small peptides penetrate tissues faster and more effectively because they have a significantly lower molecular weight and can distinguish between extracellular and intracellular domains of proteins, which antibodies cannot.

[0009] In addition, with antibodies and other macromolecules (M w Unlike antibodies (~150 kDa), small molecules are rapidly cleared from circulation, thus achieving a suitable tumor / blood ratio for imaging at an early time point after administration. This, in turn, allows physicians to obtain diagnostic information much faster compared to the use of antibody-based imaging agents.

[0010] In particular, the development of novel small imaging probes that bind to the HER2 receptor could be of great value in providing specific imaging agents for patient stratification, for predicting or monitoring response to specific anticancer therapies, and even in image-guided surgery of tumors using optical imaging, magnetic resonance imaging, nuclear imaging, computed tomography, ultrasound, and multimodal imaging techniques.

[0011] In particular, within the antibody mimicry molecule class, avidin, a compound composed of small single-chain affinity proteins (7 kDa, typically 66 amino acids), is being investigated for its high tissue penetration potential. Known examples of this type of peptide are... (Affilogic SAS, France). These peptides are derived from the ultrastable DNA-binding protein Sac7d of the thermophilic archaea *Sulfolobus acidocaldarius*, from which they retain most of the favorable biophysical characteristics, such as tolerance to temperature (up to 90°C) and pH (0–13). In particular, high-affinity avidins are engineered using several rounds of ribosome display via complete randomization of 10–14 amino acid residues at the DNA-binding site of Sac7d, and can be tuned to have high affinity for specific targets expressed on the surface of cancer cells while retaining the original thermophilic and acidophilic stability characteristics. 2,3 .

[0012] In addition to being extremely stable and robust, avidin peptides offer better pharmacokinetic profiles for antibody imaging and can be easily mass-produced using recombinant bacterial technology.

[0013] For example, Goux M. et al., Bioconjugated Chem. 2017, 28, 2361-2371. 4 It was made public when it was used 18 When radiolabeling with F-FBEM, anti-EGFR nanofitin B10 is used as an imaging agent, particularly as a radiotracer for targeted PET. However, the specific sequence of the avidin that binds to the target HER2 has not been described.

[0014] Among known antibody mimic peptides, several affinity sequences for HER2 binding have been investigated, such as those in WO2009 / 080810. 5 The name Affibody AB is used in Orlova A. et al., Cancer Res. 2006, 66(8), 4339-4348. 6 As described in [the text].

[0015] In WO2012 / 096760 7 Examples of using such HER2-binding peptides as imaging agents after conjugation with radionuclides and chelating agents have been reported by GE Healthcare Ltd and Affibody AB.

[0016] WO2017 / 161096 8 The company Tarveda Therapeutics Inc. discloses nanoparticles and microparticles containing generic conjugates with targeting moieties (such as Nanofitin), linkers, and activators, although no specific examples of such conjugates have been reported.

[0017] Therefore, despite efforts, there is still a need to identify and develop HER2 binding agents as described above, characterized by high and specific affinity for the target, for use as diagnostic agents for HER2-positive tumors, and particularly for monitoring the treatment of HER2-positive tumors. Summary of the Invention

[0018] This invention relates to a novel polypeptide sequence characterized by an avidin structure that specifically targets the HER2 receptor with high affinity.

[0019] Specifically, this invention relates to HER2-binding peptides comprising the following amino acid sequence:

[0020] VKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREK (SEQ ID NO: 1).

[0021] Given that HER2 protein overexpression is a marker of pathological conditions associated with breast cancer and many other types of tumors, the HER2-binding avidin of the present invention, once labeled with imaging components, could be used for early cancer diagnosis and staging from the perspective of more effective therapeutic treatment and / or reducing patient overtreatment.

[0022] Therefore, they can be effectively conjugated to labeled portions, enabling easy identification and evaluation during in vitro and / or in vivo analysis. Such conjugates or complexes are also an object of this invention.

[0023] The avidin of the present invention has been found to possess properties suitable for use in diagnostic tools, such as nanomolar affinity for the target HER2, non-binding to albumin, low predictive immunogenicity, good internalization in HER2-positive cells, and good expression yield. In particular, the avidin and its conjugates of the present invention can specifically recognize the target at physiologically present sites (i.e., on the cell surface expressing HER2), thus allowing for in vivo application.

[0024] Furthermore, it has been discovered that the avidin of the present invention can recognize specific epitopes of HER2 that are different from those recognized by trastuzumab and pertuzumab, monoclonal antibodies currently used in the clinical treatment of HER2-positive tumors. This allows, for example, the avidin of the present invention to be used as a reagent to test changes in HER2 receptor expression in response to trastuzumab and / or pertuzumab therapy. Attached Figure Description

[0025] The features of the invention can be better understood by referring to the following detailed description and accompanying drawings, wherein:

[0026] Figure 1 The RP-HPLC chromatogram of the Aff04 peptide shows its presence in a dimer form;

[0027] Figure 2 The MALDI-TOF mass spectra of the Aff04 peptide after disulfide bond reduction and alkylation (acquisition range 600-20,000 m / z) are shown.

[0028] Figure 3 The affinity of the conjugate IRDye80iCW-Aff04 for the HER2-positive cell line SKBR3 relative to the HER2-negative cell line A431 was reported.

[0029] Figure 4Results of competitive ELISA assays using trastuzumab-biotin or pertuzumab-biotin relative to Aff04 peptide were reported.

[0030] Figure 5 Results of different competitive ELISA assays using trastuzumab-biotin relative to Aff04 peptide (a) or pertuzumab-biotin relative to Aff04 peptide (b) are reported.

[0031] Figure 6 The curves for trastuzumab (a) or pertuzumab (b) in the presence or absence of Aff04 were reported;

[0032] Figure 7 The fluorescence signal curves (mean, standard deviation, n=3) of the conjugate IRDye800CW-Aff04 administered at a dose of 10 nmol / mouse in Balb / c nu / nu mice are reported.

[0033] Figure 8 The in vitro biodistribution of the conjugate IRDye800CW-Aff04 in healthy mice was reported 48 hours after injection at a dose of 10 nmol / mouse.

[0034] A brief explanation of sequence listings

[0035] -SEQ ID NO: 1 lists the 64aac sequence of the avidin named Aff03 of this invention:

[0036] VKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREK;

[0037] -SEQ ID NO: 2 lists the 77aac sequence of the derivative variant corresponding to SEQ ID NO: 1, which was engineered with a Cys tag at the C-terminus and a hexahistine tag at the N-terminus and named avidin Aff04:

[0038] MRGSHHHHHHGSVKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREKC;

[0039] -SEQ ID NO: 3 identified His6-tag derivatives linked to the N-terminus of avidin Aff04 and Aff04-0:

[0040] MRGSHHHHHHGS;

[0041] -SEQ ID NO: 4 is an engineered polypeptide corresponding to Aff04 (SEQ ID NO: 2), which does not contain a C-terminal cysteine ​​residue and is named Aff04-0:

[0042] MRGSHHHHHHGSVKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREK;

[0043] -SEQ ID NO: 5 identified an engineered polypeptide formed by fusing the sequence Aff04-0 (SEQ ID NO: 4) with a truncated portion of Pseudomonas exotoxin A (Pe38):

[0044] MRGSHHHHHHGSVKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREKKLGSAGSAAGSGEFGGSLAALTAHQACHLPLE TFTRHRQPRGWEQLEQCGYPVQRLVALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALTLAAAESERFVRQGTGNDEAGAASGPADSGDALLERNYP TGAEFLGDGGDVSFSTRGTQNWTVERLLQAHRQLEERGYVFVGYHGTFLEAAQSIVFGGVRARSQDLDAIWRGFYIAGDPALAYGYAQDQEPDARGRIRNGALLRVYVP RSSLPGFYRTGLTLAAPEAAGEVERLIGHPLPLRLDAITGPEEEGGRLETILGWPLAERTVVIPSAIPTDPRNVGGDLDPSSIPDKEQAISALPDYASQPGKPPKDEL. Invention Details

[0046] One object of the present invention is to provide novel agents with affinity for human epidermal growth factor receptor 2 (HER2), particularly polypeptides and their derivatives and conjugates characterized by specific binding to HER2.

[0047] Therefore, in a first aspect, the present invention provides a polypeptide that specifically binds to HER2, comprising the amino acid sequence VKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREK (SEQ ID NO: 1). Preferably, such a sequence has a length of up to 100 amino acids. More preferably, it has a length of up to 80 amino acids.

[0048] In a preferred embodiment, the present invention provides a polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 1.

[0049] Without departing from the scope of the invention, various modifications and / or additions may be made to the polypeptides defined above, including, for example, adding additional amino acids to the sequence, derivatizing with any linker or spacer, or conjugating with a specific marker or imaging motif, as described in more detail below.

[0050] For example, the present invention also includes peptides in which the aforementioned HER2-binding peptides have additional amino acid residues added at one or both terminal positions, provided that they do not alter the biological function of the peptide. These additional amino acid residues may function in HER2 binding of the peptide, but can also be well used for other purposes, such as those related to peptide production, purification, stabilization, coupling, and / or detection. Preferably, such additional amino acid residues may comprise one or more amino acid residues added for chemical coupling purposes. A preferred embodiment involves adding at least one amino acid containing a thiol group (e.g., cysteine ​​or homocysteine) or a primary amino group (e.g., lysine) or a carboxylic acid group (e.g., glutamic acid or aspartic acid) at the first or last position of the peptide chain, i.e., at the N or C terminus.

[0051] In another embodiment, such residues for chemical coupling can also be introduced by replacing another amino acid on the surface of the protein domain (preferably on a portion of the surface that does not participate in target binding).

[0052] In some embodiments, the residues suitable for coupling may also be represented by synthetic or non-natural amino acids or amino acid mimics that function similarly to naturally occurring amino acids. Non-limiting examples are selected from β-amino acids. 2 -or β 3 - Amino acids; γ-amino acids; substituted glycine or alanine, such as p-acetylphenylalanine or p-azidophenylalanine; 6-aminohexanoic acid and other derivatives known in the art, which can be used to modulate coupling strategies or impart stability to conjugates.

[0053] Additional amino acid residues may also contain “tags” for the purification, separation or detection of the peptide, such as a hexahistyl tag, or a “myc” tag or “FLAG” tag that interacts with an antibody that is specific to the tag, or other alternatives known to those skilled in the art, which may be used alone or conjugated to a binding target.

[0054] Therefore, in another preferred embodiment, the present invention provides a HER2-binding polypeptide comprising the amino acid sequence MRGSHHHHHHGSVKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREKC (SEQ ID NO: 2), corresponding to the peptide of SEQ ID NO: 1 modified by adding Cys to the C-terminus and the amino acid sequence MRGSHHHHHHGS (SEQ ID NO: 3) with a His6-tag at the N-terminus.

[0055] In a preferred embodiment, the present invention provides a HER2-binding polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 2.

[0056] In another preferred embodiment, the present invention provides a HER2-binding polypeptide comprising the amino acid sequence MRGSHHHHHHGSVKVKFGHMGEEKEVDTSKIYAVNRAGKFVHFAYDDNGKFGSGSVPEKDAPKELLDMLARAEREK (SEQ ID NO: 4), corresponding to the peptide of SEQ ID NO: 1 modified by adding the amino acid sequence MRGSHHHHHHGS (SEQ ID NO: 3) with a His6-tagged label to the N-terminus.

[0057] In another embodiment, the present invention provides a HER2-binding polypeptide consisting of an amino acid sequence as shown in SEQ ID NO: 4.

[0058] In particular, the additional six-histidine tag at the N-terminus of the peptide can be used to purify the protein via a Ni-NTA column according to known procedures, such as Bornhorst JA et al., Methods Enzymol. 2000, 326, 245-254. 9 As described in [the text].

[0059] The present invention also covers multimers, such as dimers, of polypeptides comprising the sequence SEQ ID NO: 1. For example, in one embodiment, the present invention provides a homodimer molecule comprising two polypeptides comprising the amino acid sequence SEQ ID NO: 1 or a heterodimer molecule comprising a polypeptide comprising the amino acid sequence SEQ ID NO: 1 and a different polypeptide having a high binding affinity for HER2 or other target molecules, to produce multispecific reagents that can be used for a variety of biotechnological applications.

[0060] The linked polypeptide “units” in such polymers according to the invention can be covalently linked using known organic chemical methods, or expressed as one or more fusion polypeptides in a system for recombinant polypeptide expression, or linked directly or through a linker such as an amino acid linker in any other way.

[0061] In a variety of applications, all the peptides defined above can be considered suitable alternatives to antibodies against HER2. Therefore, one aspect of the invention relates to HER2-binding peptides as described above, which are conjugated to or labeled with compounds to form a reporter motif, suitable for the diagnosis or imaging of cancerous diseases caused by and / or associated with HER2 overexpression.

[0062] The “report portion” refers to a molecule that can be detected directly or indirectly by imaging techniques, wherein the HER2-binding peptide of the present invention is linked to at least one detectable label or material (e.g., a dye whose optical properties can be measured; a contrast agent containing magnetic particles; or a gas containing vesicles). For example, the report portion is not directly detectable when it becomes detectable only through interaction with the environment or other materials that alter its detectability.

[0063] Suitable imaging techniques for detecting such report sections include, for example, magnetic resonance imaging (MRI), positron emission tomography (PET), computed tomography (CT), ultrasound (US), photoacoustic imaging (PAI), near-infrared fluorescence (NIRF), and single-photon emission computed tomography (SPECT), or techniques related to optical imaging (OI).

[0064] In particular, PET imaging technology also includes immunoPET, in which disease-specific biomarker information is obtained by directly targeting receptors of interest (e.g., with antibodies or antibody-mimicking molecules).

[0065] For imaging applications, the peptide of the present invention is linked to a label typically selected from the following: a fluorophore moiety capable of generating a fluorescent signal, such as fluorescein, FITC, Alexa dye, cyanine dye, DyLight dye, IRDye dye, or VivoTag dye; an optical moiety comprising an agent that can be used to generate contrast or a signal using optical imaging; a magnetic or paramagnetic moiety comprising a chelating agent for magnetic resonance capable of forming a stable complex with paramagnetic metal ions, such as Gd(III), Mn(II), Cr(III), Cu(II), Fe(III), Pr(III), Nd(III), Sm(III), Tb(III), Yb(III), Dy(III), Ho(III), and Er(III); and a radiolabeled isotope, including, for example, 18 F, 124 I, 11 C 64 Cu、 68 Ga、 89 Zr、 44 Sc and 99m Tc and other radioisotopes of indium, gallium, yttrium, bismuth, radioactive actinides and radioactive lanthanides; affinity tags, such as biotin; X-ray response portions that can be used to generate contrast or signals using X-ray imaging, such as chelates of iodinated organic molecules or heavy metal ions; ultrasound response portions or components for contrast-enhanced ultrasound imaging, preferably in the form of inflated microbubbles; photoacoustic response imaging portions, including photoacoustic imaging compatibilizers; and nanoparticle-based portions.

[0066] Preferably, in the case of radionuclide labeling, this complexation is carried out using a chelating agent or a polydentate ligand to form a chelate (particularly with a radioactive metal). Non-limiting examples of such chelating agents are 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA); 2,2',2”-(1,4,7-triazacyclononane-1,4,7-triyl)triacetic acid (NOTA); 2-[bis[2-[bis(carboxymethyl)amino]ethyl]amino]acetic acid (DTPA); ethylenediaminetetraacetic acid (EDTA); 10-(2-hydroxypropyl)-1,4,7-tetraazacyclododecane-1,4,7-triacetic acid (HP-DO3A); 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazazolide (AAZTA) and their derivatives.

[0067] Therefore, in a preferred embodiment, the present invention comprises a radiolabeled polypeptide consisting of a chelate of the aforementioned HER2-binding polypeptide and a radionuclide such as a radiometal. More preferably, the radiometal is 68 Ga、 44Sc or 99m Tc.

[0068] In another preferred embodiment, the polypeptide of the present invention is conjugated with a fluorophore selected from anthocyanin dyes (e.g., IRDye800CW, IRDye650, IRDye680, Cy3, Cy5, Cy5.5, Cy7, ZW800-1, AlexaFluor750, AlexaFluor790, and their analogues and derivatives).

[0069] In addition, tagging systems can be used to link peptides and labels, including biotin / avidin, biotin / streptavidin, and biotin / neutral avidin.

[0070] Therefore, in another preferred aspect, the present invention provides a polypeptide as defined above linked to an affinity marker such as biotin.

[0071] The coupling between the targeted peptides and the labeled or imaging moieties of the present invention can be performed by the methods described in the embodiments herein or other methods well known to those skilled in the art. For example, they can be covalently or non-covalently linked, optionally by inserting suitable adapters or spacers for labeling.

[0072] Typically, such connectors or spacers may contain, alone or in combination, amino acid or nucleic acid sequences or reactive motifs, including, for example, aminooxy, azide, alkynyl, thiol, or maleimide groups.

[0073] Such a linker preferably comprises two functional parts: one providing rapid and efficient labeling, and the other enabling rapid and efficient coupling with the peptide of the present invention, for example, via an amine group or preferably via a thiol group of cysteine. For example, the maleimide group reacts with the thiol group to form a stable thioether bond. Preferably, the final complex is formed by first reacting the label with the linker, and then with the thiol group of the peptide.

[0074] The peptides of this invention (appropriately conjugated with labels to form a reporter moiety) can be used for the diagnosis and staging of HER2-related states, conditions, dysfunctions, illnesses, or diseases, and for monitoring treatment responses in such states, particularly for targeting cancers characterized by HER2 protein overexpression in a clinical setting. Exemplary applications include the diagnosis of cancers associated with HER2 expression, such as breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, and gastrointestinal tumors.

[0075] Therefore, in another aspect, the present invention provides peptide conjugates as defined above for the diagnosis or visualization of cancerous diseases caused and / or associated with HER2 overexpression, i.e., for the identification or determination of the nature and cause of HER2-related diseases by assessing patient history, examining and reviewing laboratory data. In particular, such peptide conjugates can be used for imaging body tissues or organ systems that overexpress HER2. More preferably, it can be used to determine HER2 expression before and after treatment, which can be accomplished by acquiring images before and after treatment, and to determine the degree or anatomical content of HER2 expression, for example, for surgical purposes or for patient stratification.

[0076] In another aspect, the present invention provides the use of the polypeptide conjugates described above in the preparation of imaging agents for detecting cancerous tissues or organs characterized by HER2 overexpression.

[0077] In another aspect, the present invention provides a composition comprising a polypeptide or polypeptide conjugate as defined herein, and one or more suitable pharmaceutically acceptable carriers, excipients, diluents, and / or additives. The composition may vary depending on its intended use, whether for diagnostic or imaging applications. Such a composition may also contain one or more adjuvants selected from preservatives, wetting agents, emulsifiers, and dispersants.

[0078] The composition may further contain one or more different imaging agents.

[0079] In one embodiment, the present invention provides a composition as described above for imaging target tissue containing HER2, wherein the composition comprises a polypeptide of the present invention conjugated or labeled with an imaging portion as defined above. The composition may also be in the form of liposomes or nanoparticles, and may be suitable for different types of application. Preferably, the imaging is performed using an imaging technique selected from magnetic resonance imaging, positron emission tomography (PET), computed tomography (CT), ultrasound (US), photoacoustic imaging (PAI), near-infrared fluorescence (NIRF), and single-photon emission computed tomography (SPECT), or an imaging technique associated with optical imaging (OI).

[0080] In one embodiment, the composition is suitable for parenteral administration, preferably intravenous or subcutaneous administration, for example in the form of a sterile aqueous solution, dispersion, or powder for the preparation of sterile injectable solutions, dispersions, or emulsion formulations. In another embodiment, the above composition can be applied topically or by inhalation.

[0081] The composition is provided for use with imaging techniques to visualize tumors expressing HER2, such as breast cancer.

[0082] In another aspect, the present invention provides a kit containing at least one polypeptide of the present invention in one or more containers, preferably labeled or conjugated with an imaging portion, and instructions for use.

[0083] The present invention also provides a method for in vivo imaging of at least a portion of mammalian subjects, preferably humans, with cancer characterized by HER2 overexpression, the method comprising the following steps:

[0084] - Administer the composition as described above to the subject;

[0085] -Optionally monitor the delivery of the composition to the subject;

[0086] - Imaging of the subject using diagnostic equipment; and

[0087] - Subjects who are optionally diagnosed with HER-2 related disease conditions.

[0088] The polypeptides of the present invention can be obtained by recombinant expression, i.e., by sequence cloning in an expression plasmid (which can be expressed in, for example, *E. coli*) and purification by affinity chromatography, for example according to Huet S. et al., PLoS ONE 2015, 10(11):e0142304 10 The program described.

[0089] For example, the preculture can be grown overnight at 37°C in a medium containing glucose and antibiotics. The preculture can be diluted in a medium containing glucose and antibiotics and grown to mid-log at 37°C. Protein expression can then be induced by adding isopropyl β-D-1-thiogalactopyranoside, and the culture can be shaken overnight at 30°C. Bacteria can be precipitated by centrifugation and then resuspended in lysis buffer. Cell lysis can be performed at room temperature for 1 hour, and the suspension can be centrifuged to remove cell debris. The Histag protein can then be purified from the supernatant by immobilized metal ion affinity chromatography (IMAC) using nickel resin and an elution buffer containing 250 mM imidazole.

[0090] Another aspect of the present invention relates to a nucleic acid molecule encoding the aforementioned polypeptide.

[0091] In another embodiment, the present invention relates to an expression vector comprising the aforementioned nucleic acid molecule and optionally other nucleic acid elements, which enables the production of the polypeptide according to the invention through expression of the nucleic acid molecule.

[0092] Furthermore, the present invention relates to host cells (e.g., eukaryotic cells, prokaryotic cells, or plant cells) containing the expression vector.

[0093] The foregoing aspects represent recombinant technologies known to those skilled in the art for producing the polypeptides of the present invention.

[0094] Alternatively, the polypeptides of the present invention can also be produced by other known methods, including chemical synthesis, for example using standard solid-phase synthesis techniques, or expressed in different hosts such as plants and transgenic animals.

[0095] The invention will now be described in detail by way of an embodiment performed according to the invention.

[0096] Experimental Section

[0097] The following examples are provided for illustrative purposes only and should not be construed as limiting the invention.

[0098] equipment

[0099] The sequences and conjugates of the present invention prepared according to the following examples were characterized by UV / VIS (optical density), SE-HPLC, RP-HPLC, and MALDI-TOF MS analysis data using one of the following methods:

[0100] SE-HPLC: Size exclusion HPLC analysis was performed at 30 °C using a Sepax Zenix SEC-80 4.6 x 300 mm column (injection volume: 10 μL) at a flow rate of 1 mL / min. The instrument was equipped with a 280 / 780 nm UV / VIS detector. The mobile phase was 150 mM phosphate buffer, pH 7.0.

[0101] RP-HPLC: Unless otherwise specified, HPLC analyses were performed at 40 °C using a Jupiter Proteo (Phenomenex) 4.6 x 250 mm column (injection volume: 10 μL) at a flow rate of 1.2 mL / min. The instrument was equipped with a 280 / 780 nm UV / VIS detector. Mobile phase A was 0.1% TFA in water, and mobile phase B was 0.1% TFA in acetonitrile. The gradients are reported below:

[0102] MALDI-TOF MS: Mass spectra were obtained using a MALDI-TOF Ultraflex II mass spectrometer (Bruker Daltonics). Samples were pretreated by loading 10 μL pipette tips (C18 ZipTip) to remove salts and eluted with a 10 μL volume. After sample purification, mass spectra were obtained in the range of 600 to 20,000 m / z using a final sample concentration of 100 μM.

[0103] Flow cytometry analysis (FACS) using BD Accuri TMThe flow cytometry was performed using a C6 flow cytometer (BD Biosciences).

[0104] Affinity assays were performed using biolayer interference (BLI) assays with the Octet system and a protein A biosensor (FortéBio). Recombinant human protein hHER2 was purchased from R&D Systems (Minneapolis, US).

[0105] In vivo imaging experiments were conducted using the IVIS spectral in vivo imaging system (Perkin Elmer Inc.), which is equipped with 10 narrowband excitation filters (30 nm bandwidth) and 18 narrowband emission filters (20 nm bandwidth) spanning 430–850 nm.

[0106] The Aff04-AAZTA conjugate was purified by preparative HPLC on a Waters AutoPurification system equipped with a 3100 mass detector, a 600 quaternary pump gradient module, a 2767 sample manager, and a 2487 UV / Vis detector. Atlantis was used. C 18 Purification was performed using an OBD 5 μm (19 x 100 mm) column. Eluent A: 0.1% TFA in H₂O; Eluent B: 0.1% TFA in CH₃CN. Gradient curve: a linear gradient from 10% to 100% B over 1 minute at 10% B, followed by isocratic gradient at 100% B for 2 minutes. Flow rate: 20 mL / min.

[0107] The purity of the product was monitored by analytical HPLC, which used a Waters 2695 Alliance separation module equipped with a Waters 2998 photodiode array detector and employed the following:

[0108] (For AAZTA derivatives) Waters Atlantis DC18 5μm (4.6 x 150 mm) column. Gradient curves: 10% B 0 min, 10% B 5 min, 65% B 15 min, 100% B 20 min, 100% B 25 min. Flow rate: 1 mL / min; λ: 210 nm; or

[0109] (For the Aff04-AAZTA conjugate) Waters Xterra RP8 5μm, 4.6x150mm column. Gradient curves: 25% B 0 min, 25% B 5 min, 33% B 11 min, 33% B 15 min, 80% B 17 min, 95% B 18 min, 95% B 20 min.

[0110] Abbreviations

[0111] HER2 (human epidermal growth factor receptor 2)

[0112] kDa (kilodaltons)

[0113] HSA human serum albumin

[0114] MSA mouse serum albumin

[0115] BSA (Bovine Serum Albumin)

[0116] ELISA (Enzyme-Linked Immunosorbent Assay)

[0117] BLI biological layer interference

[0118] K d Dissociation equilibrium constant

[0119] aac amino acids

[0120] RT room temperature

[0121] The truncated portion of Pe38 Pseudomonas exotoxin A

[0122] TCEP tri(2-carboxyethyl)phosphine

[0123] UV / VIS ultraviolet / visible spectrophotometry

[0124] SE-HPLC size exclusion high-performance liquid chromatography

[0125] RP-HPLC (Reversed-Phase High-Performance Liquid Chromatography)

[0126] DTT dithiothreitol

[0127] MALDI-TOF matrix-assisted laser desorption / ionization-time-of-flight mass spectrometry

[0128] PBS phosphate buffer

[0129] TBS-T Tris Buffered Saline – Tween 20

[0130] TMB Tetramethylbenzidine

[0131] TCEP tri(2-carboxyethyl)phosphine

[0132] HRP (Hordeum peroxidase)

[0133] DIPEA N,N-Diisopropylethylamine

[0134] HATU 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-hexafluorophosphate oxide

[0135] TFA (trifluoroacetic acid)

[0136] TIS Triisopropylsilane

[0137] The abbreviations for individual amino acid residues are conventional: for example, Cys or C is cysteine, Asp or D is aspartic acid, Gly or G is glycine, and Arg or R is arginine. Unless otherwise stated, amino acids referred to herein should be understood to have the L-isomer configuration. Example

[0138] Example 1: Preparation of avidin according to the present invention

[0139] To selectively target the HER2 receptor, an avidin library was enriched against the target through several rounds of sequential screening (up to 6 rounds). Enrichment of anti-HER2 avidin was monitored by ELISA assays after each selection round, with conditions controlling for negligible binding to human (HSA), mouse (MSA), and bovine (BSA) serum albumin. At the end of the selection process, an avidin named Aff03 (SEQ ID NO: 1) was found to have nanomolar affinity for HER2, does not bind to albumin, and exhibits low computed immunogenicity.

[0140] The peptide Aff04 was obtained by sequencing the selected clones identified in the screening step. (According to Huet S. et al., PLoS ONE 2015, 10(11):e0142304) 10The procedure described herein involves subcloning a sequence comprising an N-terminal His6 tag (MRGSHHHHHHGS, SEQ ID NO: 3) and a C-terminal Cys tag into *E. coli* DH5αLacIq strain. Briefly, the DNA amplicon encoding SEQ ID NO: 1 is subcloned via Gibson assembly into a plasmid derived from pQE-30 (Qiagen) to encode SEQ ID NO: 2, and the ligation mixture is transformed into *E. coli* DH5αLacIq strain (Invitrogen). Clones are isolated and selected on 2xYT plates containing 100 μg / ml ampicillin and 25 μg / ml kanamycin. Plasmid construction is confirmed by Sanger sequencing. Precultures from transformed *E. coli* DH5αLacIq are grown overnight at 37°C in 2xYT medium containing 1% glucose, 100 μg / ml ampicillin, and 25 μg / ml kanamycin. Precultures were diluted 1:20 in 2xYT medium containing 0.1% glucose, 100 μg / ml ampicillin, and 25 μg / ml kanamycin, and grown to mid-log (OD600 = 0.8–1.0) at 37°C. Protein expression was then induced by adding isopropyl β-D-1-thiogalactopyranoside to a final concentration of 0.5 mM, and the cultures were incubated overnight at 30°C with shaking. Bacterial pelleting was achieved by centrifugation at 3220 g for 45 min. The cell pellet was resuspended in a pH 7.4 lysis buffer consisting of 1X BugBuster protein extraction reagent, 5 μg / ml DNase I, 20 mM Tris, 500 mM NaCl, and 25 mM imidazole. Cell lysis was performed at room temperature for 1 h, and the suspension was centrifuged at 3220 g for 45 min to remove cell debris. Histag protein was then purified from the supernatant using immobilized metal ion affinity chromatography (IMAC) with His60 Nickel Superflow resin (Clontech) and a pH 7.4 elution buffer consisting of 20 mM Tris, 500 mM NaCl, and 250 mM imidazole. Additional endotoxin removal steps were performed on samples intended for cell-based assays. First, samples were buffer-exchanged by dialysis against PBS (10 mM phosphate, 2.7 mM KCl, and 137 mM NaCl, pH 7.4; Sigma-Aldrich). Samples were then filtered on a Sartobind STIC PA anion exchanger (Sartorius). Finally, samples dialyzed against PBS were filtered through a 0.2 μm pore size Minisart hydrophilic membrane (Sartorius) and stored under sterile conditions.

[0141] The derived avidin was named Aff04 (SEQ ID NO: 2).

[0142] Example 2: Determination of intracellular infiltration of avidin Aff03

[0143] The internalization of Aff03 was assessed using a specialized cell-based assay. In short, Aff03 was labeled with SEQ ID NO: 3 (i.e., forming the sequence Aff04-0) and then fused to a truncated portion of Pseudomonas exotoxin A, wherein the internalization domain of the toxin was removed, resulting in the final peptide having the sequence shown in SEQ ID NO: 5. This truncated form (Pe38) requires internalization with Aff03 to maintain its cytotoxic activity.

[0144] Then, to test internalization, the viability of breast cancer cell lines SK-BR-3 (overexpressing the HER2 receptor) and MCF-7 (low-expressing the HER2 receptor) was measured at different nanofibrin concentrations. Dose-dependent cytotoxicity induction was observed in SK-BR-3 cells, while conversely, no significant changes were observed in MCF-7 cells. The cell death induction, triggered by Aff03-induced internalization, demonstrated the efficient transport of Pe38 via the HER2 receptor.

[0145] Example 3: Coupling of avidin Aff04 with IRDye800CW-maleimide

[0146] Purchase IRDye800CW-maleimide (Li-CorInc., USA) to conjugate the activated form of this fluorophore IRDye800CW with a reactive cysteine ​​tag at the C-terminus of avidin Aff04 (SEQ ID NO: 2).

[0147] Because the terminal free cysteine ​​residue is highly reactive, it also leads to the formation of a dimer form of the protein in the stock solution. A disulfide bond does indeed form between the Cys-terminal residues of the two monomers in the presence of oxygen. Therefore, a reduction step was performed on the Aff04 stock solution prior to conjugation. The Aff04 stock solution was reduced in phosphate buffer (0.02 M phosphate, 0.054 M KCl, 0.274 M NaCl, pH 7.4) by incubation with a mild reducing agent (TCEP). Specifically, 50 μL of 0.5 M TCEP was added to 5 mg of avidin Aff04 and incubated at room temperature for 1–16 h (preferably 1–2 h). The obtained monomeric protein was purified from the excess reducing agent using a desalting column (e.g., Zeba desalting rotary column, Thermo Scientific).

[0148] The purified Aff04 was then incubated with 5 mg of IRDye800CW-maleimide (dissolved in 500 μL DMSO) at room temperature in the dark (to avoid bleaching) for 1–3 hours, preferably 2 hours. At the end of the reaction, the conjugate named IRDye800CW-Aff04 was purified on a desalting column as described above.

[0149] Fractions containing the conjugate IRDye800CW-Aff04 were collected, and their optical density from 230 to 830 nm was measured by UV / VIS spectrophotometry and by SE-HPLC (see [reference]). Figure 1 Characterized by RP-HPLC. The concentration of the generated IRDye800CW-Aff04 was 1.71 mg / mL (molar extinction coefficient: 12051.52 M). -1 cm -1 ).

[0150] RP-HPLC analysis was also performed to examine the reduction of the dimer protein. Chromatograms of the dimer and Aff04 conjugated with IRDye800CW indicated that the reduction of the disulfide bond occurred efficiently prior to conjugation.

[0151] The effectiveness of fluorescent probes was tested on cell surfaces using flow cytometry.

[0152] Example 4: Coupling of avidin Aff04 with AAZTA chelating agent

[0153] The chelating agent 1,4-bis(carboxymethyl)-6-[bis(carboxymethyl)]amino-6-methylperhydro-1,4-diazazolide (AAZTA) was prepared according to the following reaction scheme 1:

[0154]

[0155] In summary, in a 10 mL round-bottom flask, 50 mg (tBu)4-AAZTA-C4-COOH (0.0744 mmol) was dissolved in 5 mL of dichloromethane, and DIPEA (3 eq., 0.223 mmol, 39 μL) and HATU (0.9 eq., 0.0670 mmol, 25.5 mg) were added to the reaction solution. After 10 minutes, 1.1 eq. of 1-(2-aminoethyl)maleimide hydrochloride (0.0819 mmol, 14.5 mg) was added to the resulting solution. After 1 hour, the solution was washed with water (3 x 2 mL) and brine (1 x 1 mL), dehydrated on Na2SO4, filtered, and evaporated under vacuum. The residue was dissolved in TFA / TIS 95:5 (3 mL) and stirred at room temperature for 5 hours. The crude product was precipitated in cold diethyl ether (100 mL) and purified by preparative HPLC on a Waters AutoPurification system. AAZTA-N-ethylmaleimide was separated as a homogeneous peak with a retention time of approximately 4.3 min. All fractions containing the final product were collected, evaporated, and lyophilized to obtain AAZTA-N-ethylmaleimide as a white solid. The purity of the product was monitored by analytical HPLC. RT: 7.065 min.

[0156] HPLC purity: 99%, p: 11.84 mg, overall yield: 28%.

[0157] ESI-MS (m / z): Calculated value: For C 24 H 35 N5O 11 (M / Z)+570.57 Measured value: 570.33.

[0158] Joining steps:

[0159] Under an argon atmosphere, 5 eq of TCEP was added to the Aff04 avidin solution. After 30 minutes, HPLC analysis confirmed complete reduction of the disulfide bonds. 10 eq of AAZTA-N-ethylmaleimide was added to the solution, and the reaction was stirred at room temperature for 45 minutes. HPLC analysis confirmed complete conversion to the desired product. The solution was purified twice on a Zeba rotary desalting column at 7K MWCO to separate all AAZTA-N-ethylmaleimide from the Aff04-AAZTA solution. The concentrations were determined by UV spectrophotometry at 280 nm (ε 2980 cm⁻¹). -1 M -1 The collected solution was quantified. The product was characterized by LC / MS on a Waters 2695 Alliance separation module.

[0160] HPLC purity 98%

[0161] Calculated MW: 9243.22; (M(H+)12 / 12): 771.28; Experimental MW(M(H+)12 / 12): 771.25

[0162] Example 5: Analytical Characterization

[0163] The analysis and characterization of the unconjugated avidin Aff04 were performed by MALDI-TOF mass spectrometry under the conditions described above. After pretreatment with a 10 μL pipette tip C18 ZipTip to remove salts, the sample (10 μL) was treated with 2 μL of DTT (20 mM in PBS) to reduce the sulfur bridge between the two C-terminal Cys. The reaction was incubated at 50 °C for 15 min and then cooled on ice. The obtained monomer was then alkylated to avoid further dimerization. For this purpose, 2 μL of iodoacetamide (40 mM in PBS) was added, and the reaction was incubated at room temperature for 15 min. The sample was then loaded again onto a 10 μL pipette tip C18 ZipTip for purification.

[0164] MS spectra of unconjugated peptides in the m / z range of 600 to 20,000 showed two major peaks at m / z 8677 and 17405. The first peak was associated with the monomeric peptide, and the second peak was associated with the dimer peptide, formed by the creation of sulfide bridges. Following reduction and alkylation of avidin AffO4, the contribution of the first peak increased, while the contribution of the second peak decreased, as shown in the figure. Figure 2 As shown.

[0165] Example 6: In vitro affinity determination of unlabeled avidin Aff04-0 for the HER2 receptor

[0166] The affinity of the peptide of the present invention for the target HER2 was measured by biolayer interferometry. For this purpose, the protein A biosensor (FortéBio) was coated with recombinant human HER2 (hHER2) protein. After a brief wash with PBS, the coated sensor was measured relative to different concentrations (i.e., 10000, 500, 250, 125, 62.5, 31.3, 15.6, and 0 nM) of avidin Aff04-O.

[0167] Curve fitting analysis was then performed to determine the dissociation equilibrium constant. The sensor plots were analyzed using Octet data analysis software 8.2 (FortèBio) with a 1:1 binding fitting model to determine the dissociation equilibrium constant (K). d ) and the associated binding and dissociation rate constants (k on and k off ), using R 2 Values ​​are used to assess the quality of the fit.

[0168] The obtained kinetic curves show the K values ​​associated with AffO4-O avidin in the nanomolar range. d Especially 2.2±0.1x10 -8 The Kd of M is summarized in Table 1 below.

[0169] Table 1. Dissociation equilibrium constants of the hHER2-Aff04-0 complex.

[0170]

[0171]

[0172] Example 7: In vitro affinity determination of the IRDye800CW-Aff04 conjugate with the HER2 receptor

[0173] The affinity of the IRDye800CW-Aff04 conjugate for the target HER2 was measured using biolayer interferometry. For this purpose, the protein A biosensor (FortéBio) was coated with recombinant human HER2 (hHER2) protein. After a brief wash with PBS, the coated sensor was measured relative to different concentrations (i.e., 10000, 1000, 100, 10, 1, and 0.1 ng / mL) of the IRDye800CW-Aff04 conjugate.

[0174] Steady-state analysis was performed to determine the dissociation equilibrium constant. The sensor plots were analyzed using Octet data analysis software 8.2 (FortèBio) with a 1:1 bounded fitting model to determine the dissociation equilibrium constant (K). d The resulting kinetic curves show the K values ​​associated with the IRDye800CW-Aff04 conjugate in the nanomolar range. d As shown in Table 2.

[0175] These data indicate that, compared to unlabeled Aff04-0, the coupling of the fluorophore with the C-terminal cysteine ​​residue of avidin Aff04 did not significantly alter the K-value. d .

[0176] Table 2 – Dissociation equilibrium constants of the composite IRDye800CW-Aff04 conjugate / hHER2

[0177]

[0178] Example 8: In vitro affinity of the avidin IRDye800CW-Aff04 conjugate for cells

[0179] To evaluate the binding specificity of the conjugate IRDye800CW-Aff04, flow cytometry (FACS) analysis was performed on two different types of human cancer cell lines: the SK-BR-3 breast cancer cell line overexpressing the HER2 receptor (HER2+), and the A431 epidermal-like cancer cell line not expressing the HER2 receptor (HER2-). Cells were kept on ice (4°C) throughout the procedure to assess the ability of the conjugate IRDye800CW-Aff04 to bind to the exposed receptor. Briefly, 200,000 SK-BR3 (HER2+) and A431 (HER2-) cells per sample were distributed in 50 μL of FACS buffer (PBS / 0.5% BSA / 0.1% NaN3) in 1.5 mL eppendorf tubes. The conjugate IRDye800CW-Aff04 was incubated on the cells at an increased concentration for 1 hour and 30 minutes on ice. The tested concentrations were 17, 13, 8.5, 4.25, 2.12, 1.06, 0.53, 0.26, and 0.065 μM.

[0180] After centrifugation at 450 RCF for 5 minutes at 4°C and washing three times in FACS buffer, the IRDye800CW fluorophore was detected by flow cytometry analysis using 640 nm (excitation wavelength) and an Em 670 LP (long-pass filter). Recorded fluorescence values ​​were analyzed using nonlinear regression fitting with GraphPad Prism 7 software.

[0181] The obtained kinetic curves show that the Kd associated with the conjugate IRDye800CW-Aff04 is 22.4 ± 3.0 x 10⁻⁶. -8 M indicates that the peptide Aff-04 of the present invention maintains a significant affinity for the target in the HER2+ cell line SK-BR-3, even after the conjugation step. Conversely, the conjugate IRDye800CW-Aff04 showed no binding to the HER2- cell line (A431), as... Figure 3 As shown.

[0182] Example 9: Competitive ELISA of Aff04 peptide against trastuzumab or pertuzumab

[0183] A competitive ELISA assay was developed to investigate whether the epitope of the HER2 / Neu antigen recognized by the Aff04 peptide is different from the epitope recognized by the humanized monoclonal antibodies trastuzumab and pertuzumab, which are actually used to treat HER2-positive tumors.

[0184] The first assay designed to assess the quality of the assay was a competitive ELISA between trastuzumab and trastuzumab-biotin. Briefly, Medisorp clear plastic plates (96 wells, Thermo Scientific) were coated with 2.5 μg / mL HER2 / Fc chimeric protein in TBS pH 7.4 (1 hour at room temperature). The wells were then blocked with 0.5% BSA in TBS (1 hour at room temperature).

[0185] Then, different concentrations of trastuzumab (1.6, 0.8, 0.4, 0.2, 0.1, 0.05, 0.025, and 0 μg / mL) were incubated in triplicate with 0.2 μg / mL trastuzumab-biotin in each well in TBS + 0.1% Tween 20 (TBS-T) for 1 hour at room temperature. At the end of the incubation, the plate was washed three times with 250 μL of TBS-T and incubated in TBS-T with streptavidin-HRP 1:10000 for 1 hour at room temperature. The plate was washed three more times with TBS-T and developed with TMB reagent (100 μL) for 5 minutes. The reaction was blocked with 50 μL of 2M sulfuric acid, and the reading was taken at 450 nm.

[0186] The same experiment was then performed in three alternative assays using Aff04 relative to trastuzumab-biotin or pertuzumab-biotin:

[0187] Assay a) Different concentrations of Aff04 (10000, 1000, 100, 10, 1, 0.1, and 0.01 ng / mL) were incubated in triplicate in each well with 0.2 μg / mL trastuzumab-biotin or pertuzumab-biotin. As a positive control, the same experiment was repeated using anti-RSGH TAG-HRP (1:4000) instead of streptavidin-HRP to assess the chromatic affinity of Aff04 for the HER2 receptor. Plotted on Figure 4 The ELISA results in the figure show that trastuzumab-biotin, pertuzumab-biotin, and Aff04 are all HER2 receptor binders, but the epitopes recognized by trastuzumab and pertuzumab are different from those recognized by Aff04 avidin.

[0188] Assay b) An additional competitive ELISA assay was performed in the presence of a fixed Aff04 concentration using varying concentrations of trastuzumab-biotin and pertuzumab-biotin. Briefly, Medisorp clear plastic plates (96-well, Thermo Scientific) were coated with 2.5 μg / mL Her2 / Fc chimeric protein in TBS pH 7.4 (1 h at room temperature). The wells were blocked with 0.5% BSA in TBS (1 h at room temperature). Different concentrations of trastuzumab-biotin (20000, 2000, 200, 20, 2, 0.2, 0.02, and 0 ng / mL) were then incubated in duplicate in each well with 10 μg / mL Aff04 in TBS + 0.1% Tween 20 (TBS-T) (1 h at room temperature). At the end of the incubation, the plate was washed three times with 300 μL of TBS-T and incubated in TBS-T with streptavidin-HRP 1:10000 or anti-RSGH TAG-HRP 1:4000 (1 hour at room temperature). The plate was washed three times again with TBS-T and developed with TMB reagent (100 μL) for 5 minutes. The reaction was blocked with 50 μL of 2M sulfuric acid, and the readings were taken at 450 nm. The ELISA results clearly show that when the trastuzumab-biotin or pertuzumab-biotin level increased, the curve of Aff04 detected by anti-RSGH-Tag Ab showed a negligible decrease in response (see [references] respectively). Figure 5 (a and 5b).

[0189] c) Under the same experimental conditions as the previous competitive ELISA, another competitive ELISA assay was performed using different concentrations of trastuzumab-biotin and pertuzumab-biotin against buffer (non-competitive) and Aff04 (competitive) under the same conditions.

[0190] Overall, the results showed no competition between the targeted molecules, as the curves for trastuzumab and pertuzumab were equal in the presence and absence of Aff04 avidin (see [references] respectively). Figure 6 (a and 6b).

[0191] As can be clearly seen from the figure, Aff04 retains its HER2 binding properties, and its receptor specificity is unaffected by the presence of the two reference antibodies. In fact, competitive studies have shown that the epitope recognized by Aff04 is separate and different from the epitopes recognized by trastuzumab and pertuzumab monoclonal antibodies, therefore they do not compete for HER2 binding.

[0192] Example 10: In vivo biodistribution of the conjugate IRDye800CW-Aff04

[0193] The in vivo optical imaging biodistribution of IRDye800CW-Aff04 was evaluated in healthy mice. Optical imaging experiments were performed on three healthy mice using the IVIS Spectrum system after administration of IRDye800CW-Aff04 at a dose of 10 nmol / mouse in an administration volume of 0.2 mL. Experiments were conducted at 15 min, 30 min, 1 h, 2 h, 4 h, 6 h, 24 h, and 48 h following intravenous administration of the conjugate.

[0194] Regions of interest (ROIs) were plotted in the healthy mouse reference background (hind leg muscles) and the whole body for each fluorescence image at each time point to assess signal intensity in the tissues, expressed as mean radiative efficiency (see [link to relevant documentation]). Figure 7 The fluorescence signal curves of IRDye800CW-Aff04 administered at 10 nmol / mouse in Balb / c nu / nu mice were reported.

[0195] The half-life of the test product was assessed by pharmacokinetic analysis of the signal using a single exponential decay model. The results are shown in Table 3.

[0196] Table 3 – Half-life values ​​of IRDye800CW-Aff04 in muscle and throughout the body

[0197] muscle whole body Half-life (h) 3.255 3.220

[0198] The two calculated half-life values ​​obtained are consistent, giving an average of 3.23 hours.

[0199] Such a biological half-life makes Aff04 conjugates potentially suitable as an alternative tool for rapid imaging schemes, such as optical imaging.

[0200] After euthanasia, several organs were collected for residual fluorescence measurements (48 hours later). Fluorescence signal intensity measurements were obtained from the analysis of each organ, with a region of interest (ROI) selected at the center of each sample. The organs analyzed were: kidney, lung, spleen, liver, muscle, and heart.

[0201] Figure 8 The in vitro biodistribution of IRDye800CW-Aff04 in healthy mice was shown 48 hours after injection at a dose of 10 nmol / mouse. The high renal uptake of IRDye800CW-Aff04 indicates that it is preferably cleared via the renal pathway rather than the hepatobiliary pathway.

[0202] References

[0203] 1. Slamon DJ et al., Science 1989, 244, 707-712

[0204] 2. McAfee JG et al., Biochemistry 1995, 34, 10063-10077

[0205] 3. Mouratou B. et al., PNAS 2007, 104(46), 17983-17988

[0206] 4. Goux M. et al., Bioconjugated Chem. 2017, 28, 2361-2371

[0207] 5.WO2009 / 080810A1

[0208] 6. Orlova A. et al., Cancer Res. 2006, 66(8), 4339-4348

[0209] 7.WO2012 / 096760A1

[0210] 8.WO2017 / 161096A1

[0211] 9. Bornhorst JA et al., Methods Enzymol. 2000, 326, 245-254.

[0212] 10. Huet S. et al., PLoS ONE 2015, 10(11):e0142304. sequence list <110> Borak Imaging AG <120> Anti-HER2 peptide derivatives as novel diagnostic molecular probes <130> B0723 <150> EP19217623.8 <151> 2019-12-18 <160> 5 <170> PatentIn version 3.5 <210> 1 <211> 64 <212> PRT <213> Sulfolobus acidocaldarius <400> 1 Val Lys Val Lys Phe Gly His Met Gly Glu Glu Lys Glu Val Asp Thr 1 5 10 15 Ser Lys Ile Tyr Ala Val Asn Arg Ala Gly Lys Phe Val His Phe Ala 20 25 30 Tyr Asp Asp Asn Gly Lys Phe Gly Ser Gly Ser Val Pro Glu Lys Asp 35 40 45 Ala Pro Lys Glu Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys 50 55 60 <210> 2 <211> 77 <212> PRT <213> Artificial sequence <220> <223> Engineered HER2-binding polypeptide <400> 2 Met Arg Gly Ser His His His His His His Gly Ser Val Lys Val Lys 1 5 10 15 Phe Gly His Met Gly Glu Glu Lys Glu Val Asp Thr Ser Lys Ile Tyr 20 25 30 Ala Val Asn Arg Ala Gly Lys Phe Val His Phe Ala Tyr Asp Asp Asn 35 40 45 Gly Lys Phe Gly Ser Gly Ser Val Pro Glu Lys Asp Ala Pro Lys Glu 50 55 60 Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys Cys 65 70 75 <210> 3 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Synthetic 6xHis tag derivatives <400> 3 Met Arg Gly Ser His His His His His Gly Ser 1 5 10 <210> 4 <211> 76 <212> PRT <213> Artificial sequence <220> <223> Engineered HER2-binding peptide <400> 4 Met Arg Gly Ser His His His His His Gly Ser Val Lys Val Lys 1 5 10 15 Phe Gly His Met Gly Glu Glu Lys Glu Val Asp Thr Ser Lys Ile Tyr 20 25 30 Ala Val Asn Arg Ala Gly Lys Phe Val His Phe Ala Tyr Asp Asp Asn 35 40 45 Gly Lys Phe Gly Ser Gly Ser Val Pro Glu Lys Asp Ala Pro Lys Glu 50 55 60 Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys 65 70 75 <210> 5 <211> 434 <212> PRT <213> Artificial sequence <220> <223> Engineered HER2-binding peptide <400> 5 Met Arg Gly Ser His His His His His His Gly Ser Val Lys Val Lys 1 5 10 15 Phe Gly His Met Gly Glu Glu Lys Glu Val Asp Thr Ser Lys Ile Tyr 20 25 30 Ala Val Asn Arg Ala Gly Lys Phe Val His Phe Ala Tyr Asp Asp Asn 35 40 45 Gly Lys Phe Gly Ser Gly Ser Val Pro Glu Lys Asp Ala Pro Lys Glu 50 55 60 Leu Leu Asp Met Leu Ala Arg Ala Glu Arg Glu Lys Lys Leu Gly Ser 65 70 75 80 Ala Gly Ser Ala Ala Gly Ser Gly Glu Phe Gly Gly Ser Leu Ala Ala 85 90 95 Leu Thr Ala His Gln Ala Cys His Leu Pro Leu Glu Thr Phe Thr Arg 100 105 110 His Arg Gln Pro Arg Gly Trp Glu Gln Leu Glu Gln Cys Gly Tyr Pro 115 120 125 Val Gln Arg Leu Val Ala Leu Tyr Leu Ala Ala Arg Leu Ser Trp Asn 130 135 140 Gln Val Asp Gln Val Ile Arg Asn Ala Leu Ala Ser Pro Gly Ser Gly 145 150 155 160 Gly Asp Leu Gly Glu Ala Ile Arg Glu Gln Pro Glu Gln Ala Arg Leu 165 170 175 Ala Leu Thr Leu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Gln Gly 180 185 190 Thr Gly Asn Asp Glu Ala Gly Ala Ala Ser Gly Pro Ala Asp Ser Gly 195 200 205 Asp Ala Leu Leu Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Leu Gly 210 215 220 Asp Gly Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Gln Asn Trp Thr 225 230 235 240 Val Glu Arg Leu Leu Gln Ala His Arg Gln Leu Glu Glu Arg Gly Tyr 245 250 255 Val Phe Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Gln Ser Ile 260 265 270 Val Phe Gly Gly Val Arg Ala Arg Ser Gln Asp Leu Asp Ala Ile Trp 275 280 285 Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Leu Ala Tyr Gly Tyr Ala 290 295 300 Gln Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Leu 305 310 315 320 Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr 325 330 335 Gly Leu Thr Leu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Leu 340 345 350 Ile Gly His Pro Leu Pro Leu Arg Leu Asp Ala Ile Thr Gly Pro Glu 355 360 365 Glu Glu Gly Gly Arg Leu Glu Thr Ile Leu Gly Trp Pro Leu Ala Glu 370 375 380 Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val 385 390 395 400 Gly Gly Asp Leu Asp Pro Ser Ser Ile Pro Asp Lys Glu Gln Ala Ile 405 410 415 Ser Ala Leu Pro Asp Tyr Ala Ser Gln Pro Gly Lys Pro Pro Lys Asp 420 425 430 Glu Leu

Claims

1. A polypeptide that specifically binds to human epidermal growth factor receptor 2 (HER2), comprising the amino acid sequence shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, wherein it comprises the amino acid sequence shown in SEQ ID NO:

2.

3. The polypeptide according to any one of the preceding claims, wherein it is linked to an imaging agent.

4. The polypeptide of claim 3, wherein the imaging agent is selected from fluorophore moieties, magnetic or paramagnetic moieties, radiolabeled isotopes, affinity labels, X-ray responsive moieties, ultrasound responsive moieties, photoacoustic responsive imaging moieties, and nanoparticle-based moieties.

5. The polypeptide according to claim 4, wherein the fluorophore portion is anthocyanin dye.

6. The polypeptide according to claim 4, wherein the radiolabeled isotope is selected from... 18 F, 124 I, 11 C 64 Cu、 68 Ga、 89 Zr、 44 Sc and 99m Tc and other radioactive isotopes of indium, gallium, yttrium, bismuth, radioactive actinides and radioactive lanthanides.

7. The polypeptide according to claim 4, wherein the affinity label is biotin.

8. Use of the polypeptide according to any one of claims 3 to 7 in the preparation of reagents for diagnosing or visualizing cancerous diseases caused by and / or associated with HER2 overexpression.

9. The use according to claim 8, wherein the cancer is selected from breast cancer, head and neck cancer, ovarian cancer, lung cancer, bladder cancer, and gastrointestinal tumors.

10. The use according to claim 8, wherein the diagnosis or visualization involves imaging of body tissues or organ systems expressing HER2.

11. A diagnostic or imaging composition comprising a polypeptide as defined in any one of claims 3 to 10 and a pharmaceutically acceptable excipient.

12. A diagnostic or imaging composition comprising a polypeptide as defined in any one of claims 3 to 10 and a pharmaceutically acceptable additive.

13. A diagnostic or imaging composition comprising a polypeptide as defined in any one of claims 3 to 10 and a pharmaceutically acceptable carrier or diluent.

14. Use of the composition according to any one of claims 11 to 13 in the preparation of reagents for imaging organs and tissues overexpressing HER2.

15. The use according to claim 14, wherein the imaging is based on an imaging technique selected from magnetic resonance imaging, positron emission tomography (PET), computed tomography (CT), ultrasound (US), photoacoustic imaging (PAI), near-infrared fluorescence (NIRF), and single-photon emission computed tomography (SPECT), or an imaging technique related to optical imaging (OI).

Images