WO2012000862A1 - 11c-labelled peptide for detecting a diseased tissue - Google Patents

Abstract

The use of a peptide (1) to produce an agent for detecting a diseased tissue (18) is described. In this case, the amino acid sequence of the peptide (1) derives from the amino acid sequence of a protein formed by the diseased tissue (18), and the peptide (1) binds to a human leukocyte antigen (HLA) complex (4) which is likewise formed by the diseased tissue (18). The peptide (1) also has a ¹¹C carbon atom. A radiopharmaceutical for locating a diseased tissue (18) is also described, which radiopharmaceutical comprises such a peptide (1).

Description

description

¹¹ C-labeled peptide for detection of a diseased tissue The invention relates to the use of a peptide for the manufacture ^¬ position of an agent for detecting a diseased tissue. It further relates to a radiopharmaceutical for the localization of a diseased tissue comprising such a peptide. In modern diagnostics are used to characterize

Diseases mainly used biochemical analyzes of blood, other body fluids and tissue samples. It examines the presence and amount of molecules that are typical of a particular disease. In addition to foreign substances and unphysiological metabolites also endogenous substances are detected, which are formed, for example, only in an infection by viruses or bacteria. These include, in particular, components of the immune system, in particular antibodies. Such in vitro studies can diagnose the presence of a disease, but it is not possible to determine the exact location of the diseased tissue. For this purpose, imaging techniques such as X-ray, ultrasound, and nuclear spin tomography are typically used. They can be used to localize well ectopic cell aggregates such as tumors or swellings of individual organs. However, if a diseased tissue shows no marked morphological abnormalities, or is relatively small, it can easily be overlooked in traditional studies.

The invention is therefore based on the object, an agent be ^¬ riding determine by which a diseased tissue can be specifically and regardless of its size detected. This task is achieved by the use of a peptide solved an agent for the detection of a diseased tissue. By the amino acid sequence of the peptide derived from the amino acid sequence of a protein that is formed by the diseased tissue and to a human leukocyte antigen (HLA) complex binds, which is also formed from the morbid tissue ^¬ be, the diseased tissue can be specifically pointed by ^¬ , By peptide having a ^C-11 carbon atom, a few cells may themselves be located within a diseased tissue to adhere ^¬ hand of the radioactive signal of the peptide.

The term "peptide" refers to an organic compound of at least two amino acids linked via a peptide bond. It includes both oligopeptides of up to about ten amino acids, as well as polypeptides of up to about 30 amino acids, regardless of their primary, secondary or tertiary structure. In this case, both naturally occurring and biotechnologically or synthetically produced compounds are included. The peptide used in the invention is selected so that the amino acid sequence of the peptide derived from the amino ^¬ acid sequence of a protein that is formed by the krankhaf ^¬ th tissue and binds the peptide in a HLA complex, which is also formed from the diseased tissue.

Almost all cells of the human body present peptides, which are fragments of proteins that are inside them, on their surface. Specialized cells of the immune system recognize the protein fragments and distinguish between their own and foreign origin. If a cell presents foreign molecules, it is killed and removed by the immune system. The presentation of the fragments is done by HLA complexes. The term "HLA complex" refers to a transmembrane protein, also called "major It is composed of two polypeptide chains encoded by the human leucocyte antigen, HLA complexes that bind short-chain peptides that are formed in the degradation of both home and foreign proteins in the cell and anchor them to the human leukocyte antigen Zellaußensei ^¬ te. Each HLA complex binds only specific fragments so that the interactions between a fragment and a HLA complex on the size and the amino acid sequence of the peptide are dependent. therefore, the HLA complexes specifically bind to certain peptides therefore, the cell has an. size

Number of different HLA complexes that differ in their respective binding specificity. This results in specific combinations of peptides and HLA complexes on the same cell. Because the peptide used in the invention is derived from the amino acid sequence of a protein and binds to a HLA complex, both of which are formed by the same krankhaf ^¬ th tissue pathological tissue can be detected with the peptide. The amino acid sequence of a fragment derived from a particular protein of the diseased tissue can be determined by isolating HLA fragment complexes from samples of diseased tissue. Subsequently, the bound fragments are separated from the HLA complex by means of "reversed phase HPLC" (WO 2004/085461) and sequenced using mass spectroscopic methods. Alternatively, it is also possible to predict the sequence of fragments based on the sequence of the complete protein or by computer simulation (Hiss JA et al., 2007, Walshe VA et al., 2009). The peptide is made after the sequence of the fragment and binds specifically to the corresponding HLA complex on the surface of the diseased tissue without binding to HLA complexes of other cells. Preferably, the peptide is chosen so that the bond between the peptide and the HLA complex has a linear coefficient, called kD value, of <100 nM, preferably of <10 nM, most preferably of 7.5 nM. The term "diseased tissue" refers to cells, parts of organs or whole organs that do not or not fully fulfill their physiological function. These include, for example, cells infected with viruses or bacteria, hypertrophic tissue, inflamed tissues and organs, hyperplastic and neoplastic tissue, such as ulcers, tumors and carcinomas. Diseased cells often form proteins whose expression is typical of a particular disease, for example because they are derived from the genetic material of a virus or bacterium. The cell then presents HLA complexes on their surface bind the fragments of these pro ^¬ proteins. By being derived from a disease-specific protein, the peptide specifically binds these HLA complexes and enables reliable localization of the diseased tissue.

The detection of the peptide via its radioactive Mar ^¬ kierung with a ^C-11 carbon atom. Upon decay of the ^X1 C carbon isotope, positrons, also referred to as ß ⁺ radiation, are formed. If the positrons hit an electron, they form two photons that are in one

Angle of 180 °, ie exactly in the opposite direction, remove from each other. The photons can be detected and used to calculate the position of the positron emission, or of the ¹¹ C carbon atom. , The integration of a ^C-11 carbon atom in the peptide used according to the invention allows both the presence of as well as the positi on of the peptide ^¬ detect and image. For the preparation of an inventive peptide are to be used insbeson ^¬ particular, the methods described in the patent applications DE 10 2009 035 648.7, and DE 10 2009 035 645.2 are described suitable. Furthermore, the amount of peptides, which is located at a certain point, quantified ^¬ to.

An advantage of using an ¹¹ C-labeled peptide is its structure of endogenous amino acids, making it compatible with the organism. The peptide and its a ^¬ individual amino acids are non-toxic, they can naturally metabolized, are broken down and excreted. The use of an integrated ¹¹ C carbon atom also makes it possible to prevent a radioactive foreign substance, such as fluorine, xenon, or gallium, from having to be introduced into the organism.

Another advantage of the peptide directly labeled with ^X1 C lies in the favorable signal / background ratio during the detection of the peptide. The peptide binds to the HLA Kom ^¬ plex, with which it forms a stable, difficult to access for enzymatic degradation, compound. Free, unbound

Peptides, however rapidly metabolized and excreted from the Or ^¬ organism because they are degraded rapidly by endogenous enzymes. This creates a strong and specifi ^¬ signals are available at the position of the HLA complex, and this way is minimized background signal.

In an advantageous embodiment of the invention, the peptide has about eight to about ten amino acids. The peptide binding site of the HLA complex consists of a deep cleft formed by the N-terminal ends of the two polypeptide chains. It is extremely mobile in its ^conformation , so that HLA complexes can bind molecules of different sizes. However, the binding affinity is strongest to Pep ^¬ tiden of eight to ten amino acids, so the resulting HLA-peptide complexes are particularly stable and protected against enzymatic degradation.

In an advantageous embodiment, the peptide binds to the peptide binding site of the HLA complex. Human cell h ^¬ len form a plurality of different HLA complexes that bind different types of protein fragments. In principle, HLA I and HLA II complexes are distinguished, with HLA I complexes binding, in particular, proteins which originate from the cytoplasm of the cell and HLA II complexes, those which belong to the

Membrane of the cell belong. Within these two classes, the HLA complexes are again distinguished by the sequence of their polypeptide chains. The binding specificity between an HLA complex and a particular peptide results from the binding column of the HLA complex. The other parts of the complex do not differ greatly among the different types of HLA complexes. By binding the peptide to the binding site and not interacting with other amino acid side ^{chains of} the complex, it is ensured that it binds specifically to the HLA complex of the diseased tissue. This allows background signals to be minimized.

In an advantageous embodiment of the invention, the agent is a radiopharmaceutical. The term "radiopharmaceuticals" refers to medicines containing radionuclides whose radiation is used for diagnosis and therapy. The main applications are in oncology, Kar ^¬ ogy and neurology, as well as pharmaceutical research. When radionuclides are gamma or beta rays emittieren- de nuclides, for example Xenon ^133, "technetium, gallium ^68, fluorine ¹⁸ and used. They are usually about Kom ^¬ formers such as diethylene triamine pentaacetate (DTPA) 1,4,7, 10- tetraazacyclododecane-1, 4, 7, 10-tetraacetic acid (DOTA) or ethylenediamine tetraacetate (EDTA) to mono- or polysaccharides bound. The nuclides are detected by scintigraphy, single photon emission computed tomography (SPECT) or positron emission tomography (PET), depending on the nature of their radiation. However, due to their non-physiological constituents parts, conventional radiopharmaceuticals Nebenwirkun ^¬ gen as anaphylactic or allergic reactions that cause ^¬ by a patient in Kör. The use of a peptide from endogenous amino acids significantly reduces this risk because neither the peptide itself nor its degradation products are toxic. Moreover, it is carbon, unlike Techne ^¬ consortium or xenon, one in the body occurring element that can be metabolized na ^¬ Türlich.

According to an advantageous embodiment of the invention, the C-carbon atom is a carbonyl carbon atom of an amino acid. The carbonyl groups are part of the peptide bonds between the amino acids and are located inside the peptide. This ensures that the ^ C carbon atom is not cleaved off the peptide, as would be possible with a side chain of one of the amino acids.

In a further preferred embodiment of the invention, the C-carbon atom is the carbonyl carbon atom of the N-terminal amino acid of the peptide. This embodiment is particularly preferred because the peptide immediately after the on ^¬ bring the ¹¹ C-labeled amino acid can be used. ^ C-carbon has a half-life of only about 20 Minu ^¬ th, so that the radiation dose must be chosen higher, is the more time between the synthesis of the peptide and sides ner use. If the ¹¹ C-labeling with the N-terminal amino acid and thus in the last step of the synthesis is applied, the peptide can be used immediately after its synthesis. In this way, the time Zvi ^¬ rule the processing of ^ C-carbon and the use of the peptide is reduced so that the radiation loss during the preparation of the peptide is minimized. Therefore, the radiation dose that must be used in the processing of the ¹¹ C carbon to ensure a certain radiation intensity of the product, be correspondingly lower. The production is more cost-effective and thereby the radiation exposure for the technical staff that forth ^¬ represents the peptide reduced. In an advantageous embodiment of the invention, the peptide has at least one D-amino acid. With the exception of glycine, all amino acids have a chiral center at their alpha carbon atom and can therefore exist as configurational isomers, namely as the D or L amino acid. Endogenous peptides and proteins are largely composed of amino acids in L configuration. In addition, most natural proteases and peptidases work stereoselectively and

metabolize mainly L-amino acids. Therefore, the degradation of D-amino acids by endogenous enzymes takes longer than that of L-amino acids. This fact can be used to determine the half-life of a protein or peptide to ver ^¬ lengthen by even D-amino acids are used in addition to L-amino acids (Neundorf I et al., 2008). As a result, the pharmacological clearance, ie the time until the peptide has been eliminated from the organism, can be positively influenced. However, when replacing single L-amino acids with their D-configuration, care must be taken not to alter the binding specificity of the peptide. Another possible ^¬ ness, to influence the pharmacological clearance of the peptide, is to replace individual amino acids of the peptide by unnatural amino acids with similar chemical properties. The non-natural amino acids are metabolized more slowly because the body's own proteolytic enzymes are specifically involved in the breakdown of natural amines. Nosäuren are adjusted. In the modification of the peptide the unnatural amino acids should be chosen, however, that the binding affinity of the peptide is not changed ^¬ changed. In addition, other chemical modifications of individual amino acids of the peptide are possible in order to obtain the

To selectively influence the half-life of the peptide. Example ^¬ as can be Replace the terminal amino group of the peptide by a isonitrile. Such modification redu ^¬ the sheet, conveyed from the amino group, interaction with proteolytic enzymes without altering the bond between the peptide used in the invention and the antibody.

Another object of the invention is a radiopharmaceutical for the localization of a diseased tissue comprising a peptide having an ¹¹ C carbon atom. The amino acid sequence of the peptide originates from the amino acid sequence of Pro ^¬ teins from which is formed by the pathological tissue, and the peptide binds to a human leukocyte antigen (HLA) complex, which is also formed from the diseased tissue. As a result, only a few cells of a diseased tissue can be specifically detected with the radiopharmaceutical itself. Due to the advantages of the contained peptide, the radiopharmaceutical ^{according to the} invention provides a sensitive and specific agent for determining the position of a diseased tissue in vivo. The radiopharmaceutical is administered to the patient, and the peptides contained therein are rapidly and efficiently distributed in the body because of their size. You'm ^¬ according to the chemical equilibrium with the zellei ^¬ antigenic peptide to the HLA complex of the diseased tissue and accumulate on the surface. This tissue can at ^¬ play, a focus of inflammation by viruses or bacteria be infected cells or a tumor. The accumulation of radioactively labeled peptides is detected by positron emission tomography (PET), which determines the exact position of the infected cells, the inflammation or the tumor in the patient's body.

Similarly, healthy cells that express a spe cial ^¬ protein can be detected. For this purpose, the peptide used according to the invention is selected so that its amino acid sequence is derived from a specific, naturally formed protein. The peptide then binds to the cells that make up this protein because the cells present corresponding HLA complexes on their surface. By Markie ^¬ tion of the peptide with a ^C-11 carbon atom (PET) can be shown by means of positron emission tomography to which cells of the body, the peptide has bound.

According to an advantageous development, the ¹¹ C-carbon atom is a carbonyl carbon atom of an amino acid, preferably the carbonyl carbon atom of the N-terminal amino acid of the peptide.

In a preferred embodiment, the radiopharmaceutical is a PET biomarker. PET is an established method for detecting the radiation of radioactive elements and determining their position (Massoud TF, Gambhir SS, 2003). With the aid of detector devices arranged annularly around the patient, sectional images are created on which the decay events in their spatial distribution in the interior of the body are represented. The PET also makes it possible to determine the amount of mar ^¬-labeled molecules quantitatively in a tissue.

In addition, a method for localizing a diseased tissue in an organism is disclosed, comprising Steps a) providing a peptide, b) administering the peptide to the organism, c) detecting the peptide in the organism by positron emission tomography (PET). The amino acid sequence of the peptide is derived from the amino acid sequence of a protein formed by the diseased tissue and the peptide binds to a human leukocyte antigen (HLA) complex formed by the diseased tissue. Furthermore, the peptide has an ¹¹ C carbon atom.

With the peptide used in the invention is an HLA Kom ^¬ plex is detected inside an organism, and isolated, so that the distribution of HLA complex may be observed in the body of a Pati ^¬ ducks. In this way, for example, the size or extent of an infection or a

Tumors are determined. The peptide used according to the invention is therefore outstandingly suitable for observing the course and success of a treatment, so-called therapy monitoring.

Hereinafter, preferred embodiments of the invention will be explained with reference to the accompanying schematic drawings. FIG. 1 shows schematically the binding between a peptide 1 and a human leukocyte antigen (HLA) complex 4, which is arranged on the surface of a diseased tissue 18.

Peptide 1 comprises nine amino acids 2, of which the N-terminal amino acid 3 is radioactively labeled with an ¹¹ C carbon atom. The radioactive label is represented by an asterisk (*). The peptide 1 is arranged in the peptide binding site 5 of the HLA complex 4. The peptide binding site 5 is formed from two highly variable domains, whereby a specific affinity between the HLA complex 4 and the peptide 1 arises. The HLA complex 4 is an integra ^¬ les membrane protein that extends through a cell membrane 6 of the cells of the diseased tissue 18 therethrough. It has an extracellular 7 and an intracellular 8 area.

The peptide binding site is at the 5 extrazellulä ^¬ ren region 7 of the HLA complex 4. The membrane 6 is shown gray shaded. The ¹¹ C-labeled peptide 1 binds specifically to the free

Peptide binding site 5 of the HLA complex 4, but not to other ^¬ molecules. The peptide 1 can be used to detect the HLA Kom ^¬ plexes 4 by the positrons emitted at the decay of the ^X1 C carbon atoms are detected by positron emission tomography (PET). The location of the positron emission corresponding to the location of the peptide 1 and the bound thereto HLA complex 4. Makes a pathological Ge ^¬ weave 18 the HLA complex 4, it can be detek- advantage by the peptide. 1

For the localization of a diseased tissue 18, for example of a tumor as part of a diagnosis of cancer is administered to a Pati ^¬ ducks a radiopharmaceutical containing the ^d e ^¬ labeled peptide. 1 Peptide 1 binds specifically to HLA complex 4, which is formed by the cells of tumor 18. Since the peptide 1 ^¬ by accumulates at the tumor 18. These Anhäu ^¬ Fung is visualized by PET and so the distribution of HLA complex 4 or the position of the tumor 18 in the body of the patient determined. In this way, newly formed metastases, which form the HLA complex 4, can be detected using PET. In addition, the information obtained by the visualization of the tumor 18 may serve to medicate a tumor therapeutic, for example, amount of drug and Administration schedule to adjust according to the position, size and distribution of the tumor 18.

FIG. 2 shows a representation of a peptide 1 by means of a chemical formula.

Peptide 1 comprises nine amino acids 2 of the following sequence: glycine-valine-leucine-proline-alanine-leucine-proline-glutamine-valine.

The N-terminal glycine is by structural formula represents ^¬ Darge, the following amino acids 2 by their respective three-letter code. The sequence of the peptide is also given in SEQ ID NO: 1. The carbonyl carbon atom of the N-terminal glycine is an ¹¹ C carbon atom represented by the number 11 above the carbonyl carbon atom.

Peptide 1 is prepared by conventional protein synthesis methods and the ¹¹ C-labeled N-terminal amino acid 3 is added in the last step, because the half-life of the ^X1 carbon carbon isotope is only about 20 minutes. By peptide synthesis with the ¹¹ C-labeled amino acid is abgeschlos ^¬ sen that Peptide 1 can be used immediately after labeling.

The peptide of the sequence SEQ ID NO: 1 is derived from the human glycoprotein chorionic gonadotropin (hCG-beta) (SEQ ID NO: 2), which fulfills the functions of a hormone during pregnancy. It influences the development of the embryo, in particular the differentiation of trophoblasts and embryonic blood vessel formation. In addition, however, hCG-beta is also produced by cells of various tumor types, such as breast, liver and lung tumors. The hCG-beta is degraded by the tumor cells into shorter peptides and in Form of complexes of HLA and hCG-beta peptides presented on the cell surface. In this case, the peptide of the SEQ ID No .: 1 in the peptide binding site 5 of the HLA complex is 4 ge ^¬ prevented and the overall complex on the cell membrane of the tumor cells anchored. As a result, HLA complexes 4 having a specific affinity for the peptide of SEQ ID NO: 1 are located on the tumor 18 so that it can be detected with the ¹¹ C-labeled peptide of SEQ ID NO: 1. Figure 3 shows a schematic representation (greatly simplified by Faller A, Schünke M, The Human Body, Thieme, 2008) of a circulatory system 10 of an organism and the distribution of a peptide 1 therein. The circulation system 10 includes various organs schematically represented, such as the lungs 12, heart 13, liver 14, 15 intestine and kidney 16 and the main wires 11 which these organs ver ^¬ bind. The peptide 1 is represented by triangles along the wires 11. The degradation products 17 of the peptide 1 are represented by individual lines within the outline of the kidney 16 Darge ^¬ . Left of center of the circulatory system 10 is additionally ^¬ a diseased tissue 18, for example, a tumor or an inflammation, shown, the HLA complexes 4 carries are in turn attached to the peptides. 1

The distribution of the peptide 1 in the circulatory system 10 comprises four phases, which are listed along the top-down view. Phase I: Peptide 1 is injected into the circulatory system 10 of the organism. Phase II: is via the blood circulatory system 10 Peptide 1 in the organs 12, 13, 14, 15, and 16 of the body transported ^¬ advantage. Phase III: The circulating peptide 1 specifically binds to the HLA complexes 4, and accumulates at the morbid tissue ^¬ be 18 because this is the HLA complex. 4

Phase IV: Unbound peptide 1 is rapidly metabolised and enzymatically degraded. The organism not failed ^¬ det between own peptides and the peptide 1, because it is composed of amino acids 2, 3, which correspond to the body's own molecules. The degradation products 17 of the peptide of amino acids 1 and 2, 3 collect predominantly they are over the bladder and the ureter excreted ^¬ in the kidney 16 from where.

References: WO 2004/085461 Faller A, Schünke M; The body of man; Thieme-Verlag; 2008

Hiss JA, Bredenbeck A, Losch FO, Wrede P, Waiden P, Schneider G; Design of MHC I stabilizing peptides by agent-based explosion of sequence space; Protein Eng Des Sei. 2007

Mar; 20 (3): 99-108.

Massoud TF, Gambhir SS; Molecular imaging in living subjects: seeing fundamental biological processes in a new light; Genes Dev. 2003 Mar 1; 17 (5): 545-80.

Neundorf I, Rennert R, Franke J, Közle I, Bergmann R; Detailed analysis concerning the biodistribution and metabolism of human calcitonin-derived cell-penetrating peptides; Bioconjug Chem. 2008 Aug; 19 (8): 1596-603.

Walshe VA, Hattotuwagama CK, Doytchinova IA, Wong M,

Macdonald IK, Mulder A, Claas FH, Pellegrino P, Turner J,

Williams I, Turnbull EL, Borrow P, Flower DR; Integrating in silico and in vitro analysis of peptide binding affinity to HLA Cw * 0102: a bioinformatic approach to the prediction of new epitopes; PLoS One, 2009 Nov 30; 4 (II): e8095.

Claims

Use of a peptide (1) for the production of an agent for the detection of a diseased tissue (18), characterized in that a) the amino acid sequence of the peptide (1) is derived from the amino acid sequence of a protein formed by the diseased tissue (18), b) the peptide (1) binds to a human leukocyte antigen (HLA) complex (4), which is formed by the diseased tissue (18), and c) the peptide (1) has an ¹¹ C carbon atom. Use according to claim 1, characterized, the peptide (1) has about eight to about ten amino acids (2). Use according to claim 1 or 2, characterized, the peptide (1) binds to the peptide binding site (5) of the HLA complex (4). Use according to one of the preceding claims, characterized that the agent is a radiopharmaceutical. Use according to one of the preceding claims, characterized the ¹¹ C carbon atom is the carbonyl carbon atom of an amino acid (2), preferably of the N-terminal amino acid (3) of the peptide (1). 6. radiopharmaceutical for the localization of a diseased tissue (18) comprising a peptide (1),  characterized, in that the amino acid sequence of the peptide (1) is derived from the amino acid sequence of a protein formed by the diseased tissue (18) which binds peptide (1) to a human leukocyte antigen (HLA) complex (4) which is isolated from the diseased tissue ( 18), and the peptide (1) has an ¹¹ C carbon atom. Radiopharmaceutical according to claim 6,  characterized, the ¹¹ C carbon atom is the carbonyl carbon atom of an amino acid (2), preferably of the N-terminal amino acid (3) of the peptide (1). Radiopharmaceutical according to claim 6 or 7,  characterized,  that it is a positron emission tomography (PET) biomarker.