WO2012000843A1 - 11c-labelled peptide for detecting an antigen - Google Patents

Abstract

There is described the use of a peptide (1) for the preparation of an agent for detecting an antigen (4) which (a) includes an amino acid sequence of a paratope (7) of an antibody (6) which is directed against the antigen (4), b) binds specifically to an epitope (5) of the antigen (4) and c) includes an ¹¹C carbon atom. There is furthermore provided a radiopharmaceutical for locating a tumour, which radiopharmaceutical comprises a peptide (1) which includes an ¹¹C carbon atom. The peptide (1) includes an amino acid sequence of a paratope (7) of an antibody (6) which is directed against the tumour (18).

Description

¹¹ C-labeled peptide for the detection of an antigen

The invention relates to the use of a peptide for the production of an agent for the detection of an antigen. It also relates to a radiopharmaceutical for the localization of a tumor comprising such a peptide.

The immune system serves to ward off foreign molecules, viruses and bacteria and identifies foreign substances with the help of antibodies. These recognize and bind foreign body molecules, so-called antigens, which are then broken down by other components of the immune system and excreted. However, antibodies do not bind the entire antigen, but only small well-defined regions thereof, which are also referred to as epitopes. Each antibody has two identical antigen-binding parts called paratopes that bind to identical epitopes. The bond itself is based on electrostatic, hydrophobic and / or van der Waals interactions and / or on hydrogen bonds.

The specific binding affinities between antibody and antigen are also used for diagnostic and therapeutic purposes. Diseased cells often produce antigens, for example proteins that do not occur in healthy tissue or only in very small quantities. Tumor cells also form specific molecules, so-called tumor antigens. In order to characterize diseases, antigens, predominantly with biotechnologically produced antibodies, are detected in blood or tissue samples in vitro. For this purpose, in a second step, the resulting antibody-antigen complexes, for example via fluorescence-labeled secondary antibodies detected. However, such detection of antibody-antigen complexes is significantly more difficult in vivo. Therefore, antibodies detected in vivo are directly labeled, for example by a radionuclide. A drug containing such directly labeled antibody is WX-G250, Redectanee, from Wilex AG, Munich, Germany (Divgl CR et al., 2007). The antibody is labeled with ¹²⁴ iodine and can be detected by its radioactive radiation in the living organism. The investigation of a diseased tissue, in vivo, with the help of a complete antibody molecule, however, has considerable disadvantages. The production of directly labeled antibodies is very complicated, because they must first be produced biotechnologically and then provided with a label. As a result, their production is expensive. In addition, because of their size, antibodies in the organism are only metabolized very slowly, so that their biological half-life is about 200 hours. This significantly affects the compatibility of a radioactive drug, because the radioactive element, also referred to as radioisotope, remains in the body for a very long time. In addition, the antibodies that are not bound to an antigen continue to circulate in the organism and generate a strong background signal. To reduce the background signal, it is therefore necessary to wait until the free radiolabelled antibodies have been degraded. However, in order to be able to detect the antibodies after the long waiting time, radioisotopes with particularly long half-lives must be used. However, these emit a lower amount of radiation per time interval, so that a correspondingly high radiation dose must be used in order to ensure the necessary radiation intensity during the recording. This increases the radiation exposure for the patient. In addition, there are long examination and monitoring times of the patient.

The invention is therefore based on the object to provide a cost-effective to manufacture and tolerated by the patient agent for the detection of an antigen in vivo. This object is achieved by the use of a peptide for the production of an agent for the detection of an antigen. In that the peptide has an amino acid sequence of a paratope of a Having antibody which is directed against the antigen and specifically binds to an epitope of the antigen, the distribution and position of the antigen can be determined in an organism with this peptide. The detection of the peptide and the antigen bound thereto is made possible by the ¹¹ C carbon atom 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. Both naturally occurring and biotechnologically or synthetically produced compounds are included.

The term "antigen" refers to any type of inorganic or organic compound, in particular proteins, peptides and polysaccharides whose surface structure is suitable for being recognized and bound by an antibody. The term "antibody" denotes natural or synthetic protein complexes from the group of immunoglobulins. They are usually composed of two light and two heavy polypeptide chains, each one light chain and the N-terminal part of a heavy chain together forming a paratope of the antibody. The paratope mediates binding between the antibody and the antigen by interacting with a particular, relatively small, structure of the antigen, the epitope. The specificity of the binding is determined by the individual structure of the paratope and the epitope. For a defined epitope, the specificity of the corresponding paratope results from its amino acid sequence.

In the context of the invention, a peptide is used which binds specifically to an epitope of an antigen in order to detect this antigen. For this purpose, the peptide sos is chosen such that its amino acid sequence corresponds to the amino acid sequence of the paratope. speaks, which binds the epitope of the antigen. For this purpose, an antibody is selected which is directed against the antigen to be detected, the paratope thus interacts with an epitope of the antigen. Subsequently, the amino acid sequence of the paratope of this antibody is determined. For this it is necessary to investigate which amino acids of the polypeptide chains of the antibody are involved in the formation of the paratope. The number and type of amino acids of the paratope distinguishes antibodies from each other and determines their specificity for an epitope. The sequence of the amino acids that actually constitute the paratope can be determined, for example, by a point mutation analysis of the antibody (Colbi DW et al., 2004). For this purpose, the binding affinity of different variants of the antibody carrying different point mutations is examined. Alternatively, the paratope may also be analyzed by the use of alanine scanning and surface plasmon resonance techniques as described in Heap CJ et al., 2005. The peptide is synthesized according to the amino acid sequence of the paratope so that it has a particularly high specificity for the epitope of the antigen and does not bind to other molecules. Preferably, the peptide is chosen such that the bond between the peptide and the antigen has a linear coefficient, so-called kD value, of £ 100 nM, preferably of ≤ 10 nM, most preferably of 7.5 nM. Using such a peptide, the antigen can be detected.

The detection takes place via the radioactive labeling of the peptide with an ¹¹ C-Kohlenstoffatora. Upon decay of the ¹¹ C carbon isotope, positrons, also known as ß * -

Radiation be formed formed. When the positrons hit an electron, they form two photons, which move away from each other at an angle of 180 °, ie exactly in the opposite direction. 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 an ¹¹ C carbon atom into the peptide used according to the invention, allows both the presence and the position of the peptide to be detected and displayed. For the preparation of a peptide to be used according to the invention, the processes described in patent applications DE 10 2009 035 648.7 and DE 10 2009 035 645.2 are particularly suitable. Furthermore, the amount of peptides located at a particular site can also be quantified.

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 individual amino acids are non-toxic, they can of course be metabolized, degraded and excreted. By using a built-in ¹¹ C carbon atom can also be avoided that a radioactive impurity such as fluorine ^{l8, 133} Xenon, or "Gallium has to be introduced into the organism.

Another advantage of using a peptide labeled directly with ¹¹ C lies in the favorable signal / background

Ratio during the detection of the peptide. The peptide binds to the epitope of the antigen, with which it forms a stable complex, which is difficult to access for enzymatic degradation. Free, unbound peptides, however, become fast

metabolized and excreted from the organism, because they can be rapidly degraded by endogenous enzymes. This creates a strong and specific signal at the antigen's position, and the background signal is 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 most important fields of application are oncology, cardiology and neurology as well as drug research. As radionuclides, gamma or beta rays are emitted. de nuclides, for example, ^l33 xenon, "" technetium, "gallium, and ¹⁸ fluorine, are commonly used via complexing agents, such as diethylenetriaminepentaacetate (DTPA), 1,4,7,10-tetraazacyclododecane-1,4,7,10, Tetraacetic acid (DOTA) or ethylenediaminetetraacetate (EDTA) bound to mono- or polysaccharides 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 nonphysiological components, conventional radiopharmaceuticals can cause side effects, such as anaphylactic or allergic reactions, in a patient's body.The use of a peptide from the body's own amino acids significantly reduces this risk because neither the peptide itself nor its degradation products are toxic Carbon, in contrast to technetium or xenon, an element occurring in the body that can be naturally metabolized.

According to a preferred embodiment of the invention, the antigen is formed by a tumor. The term "tumor" refers to a local increase in the volume of a tissue, such as by an inflammatory swelling or a spontaneous, unrestrained new formation of cells. Tumor cells often express certain antigens that are recognized and bound by the body's own or biotechnologically produced antibodies. The detection of such an antigen is medically particularly relevant because it allows the detection of a tumor in vivo. According to a further preferred embodiment, the antigen is a tumor antigen. Tumor antigens are specifically expressed by tumor cells, but do not occur or only in very small amounts in healthy tissue. Tumor antigens are often proteins that play a role in the development of stem cells during embryogenesis, but are no longer shown in the adult organism. The ectopic expression of such genes leads to a particularly aggressive and fast growing form of tumors. In the context of cancer diagnosis, it is therefore particularly advantageous to detect such tumors in vivo and to determine their position. According to an advantageous development of the invention, the amino acid sequence of the peptide is the amino acid sequence of a variable region of a polypeptide chain of the antibody. In each case the formation of the paratope involves the N-terminal ends of a light and a heavy polypeptide chain of the antibody. In this area, antibodies are most different from each other because the gene coding for the polypeptide chains always changes in that region. The N-terminal ends of the polypeptide chains are therefore referred to as "variable regions". Since they determine the binding specificity of the antibody, it is advantageous to use a peptide whose amino acid sequence corresponds to the amino acid sequence of one of these regions.

According to an advantageous development of the invention, the ¹¹ 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 ¹¹ 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 ¹¹ 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 can be used directly after attachment of the ¹¹ C-labeled amino acid. ¹¹ C-carbon has a half-life of only about 20 minutes, so the higher the radiation dose, the more time is left between the synthesis of the peptide and its 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 be used. In this way, the time between the processing of the ^1- carbon and the use of the peptide is reduced, so that the radiation loss during the production 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. As a result, the production is made more cost-effective and the radiation exposure for the technical staff producing the peptide is 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 be considered as

Configuration isomers, namely as D- or L-amino acid, are present. Endogenous peptides and proteins are largely composed of amino acids in L configuration. In addition, most natural proteases and peptidases work stereoselectively and mainly metabolize 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 extend the half-life of a protein or peptide by using D-amino acids besides 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 individual L-amino acids with their D-configuration, care must be taken not to alter the binding specificity of the peptide. Another way to influence the pharmacological clearance of the peptide is to replace some of the amino acids of the peptide with non-natural amino acids having similar chemical properties. The non-natural amino acids are metabolized more slowly because the body's own proteolytic enzymes are specially adapted to the breakdown of natural amino acids. In the modification of the However, for peptides, the non-natural amino acids should be chosen so that the binding affinity of the peptide is not altered. In addition, other chemical modifications of individual amino acids of the peptide are possible to specifically influence the half-life of the peptide. For example, the terminal amino group of the peptide can be replaced by an isonitrile group. Such a modification reduces the amino group-mediated interaction with proteolytic enzymes without altering the binding between the peptide used according to the invention and the antibody.

Another object of the invention is a radiopharmaceutical for the localization of a tumor comprising a peptide having a ^U C carbon atom. By having an amino acid sequence of a paratope of an antibody directed against the tumor, the peptide can be detected in the patient's body. Due to the advantages of the peptide contained, the radiopharmaceutical of the present invention provides a cost effective and well tolerated means for determining the position of a tumor 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. They bind the epitope of the disease-specific antigen and accumulate on the tissue that produces the antigen. This tissue may be, for example, an inflammatory focus or a tumor. The accumulation of radioactively labeled peptides is detected by PET, thus determining the exact location of the inflammation or tumor in the body of the patient.

According to an advantageous development of the invention, the amino acid sequence of the peptide is the amino acid sequence of a variable region of a polypeptide chain of the antibody. 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 are represented in their spatial distribution in the interior of the curvature. PET also makes it possible to quantify the amount of labeled molecules in a tissue.

Also disclosed is a method of localizing an antigen in an organism, comprising the steps of: a) providing a peptide, b) administering the peptide to the organism, and c) detecting the peptide in the organism by positron emission tomography (PET). Here, the peptide has an amino acid sequence of a paratope of an antibody directed against the antigen, binds specifically to an epitope of the antigen, and has an ¹¹ C carbon atom.

With the peptide used according to the invention, an antigen is detected and localized inside an organism, so that the distribution of the antigen in the body of a patient can be observed. In this way, for example, the size or extent of a tumor expressing the antigen can be 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. In the following preferred embodiments of the invention will be explained with reference to the accompanying schematic drawings. Figure 1A shows schematically the binding between an antibody 6 and an antigen 4. The antibody 6 consists of two heavy polypeptide chains 9 and two light polypeptide chains 8, each represented by long and short, mutually parallel lines. One of the light polypeptide chains 8, together with the N-terminal part of one of the heavy polypeptide chains 9, in each case forms a paratope 7 of the antibody 6. One of the two identical, opposing, paratopes 7 of the antibody 6 binds to an epitope 5 of the antigen 4. The antigen 4 is located on the surface of a diseased tissue 18, which in the case shown is a tumor. FIG. 1B schematically shows peptide 1 bound to epitope 5 of antigen 4. 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 specific binding affinity between the antigen 4 and the antibody 6 is due to chemical interactions between the epitope 5 of the antigen 4 and the paratope 7 of the antibody 6. These interactions are determined by the amino acid sequences of epitope 5 and paratope 7.

The amino acid sequence of the ¹¹ C-labeled peptide 1 corresponds to the amino acid sequence of the paratope 7 of the antibody 6, so that the peptide 1 has the binding affinity of the paratope 7 and specifically binds to the epitope 5 of the antigen 4. Due to this binding specificity, the ¹¹ C-labeled peptide 1 can be used to detect antigen 4. The positrons emitted upon the decay of the ¹¹ C carbon atom are detected by positron emission tomography (PET). The location of the positron emission corresponds to the location of the peptide 1 and the antigen 4 bound thereto. Tumor cells often form substances that are absent in healthy tissue and against which natural or biotechnological antibodies 6 can be produced. The amino acid sequence of the paratopes 7 of these antibodies 6 is determined by means of point mutation analyzes (Colby DW et al., 2004). Then, an ¹¹ C-labeled peptide 1 corresponding to the amino acid sequence of the paratope 7 of the antibody 6 is prepared. It specifically binds to the epitope 5 of the tumor antigen 4. In order to determine the position of the tumor 18 in the body of a patient, the ¹¹ C-labeled peptide 1 is administered to the patient. The peptide 1 binds to the epitope 5 of the tumor antigen 4 and accumulates on the cells of the tumor 18. This accumulation is visible in a positron emission tomography (PET), so that the distribution of the tumor antigen 4 or the position of the tumor 18 be determined. In this way, newly formed metastases are detected by means of PET. Figure 2 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 circulatory system 10 includes various schematically represented organs such as lung 12, heart 13, liver 14, intestine 15 and kidney 16, and the main arteries 11 connecting these organs. The peptide 1 is represented by triangles along the wires 11. The degradation products 17 of peptide 1 are represented by individual dashes within the outline of the kidney 16. In addition, to the left of the middle of the blood circulation system 10, a pathological tissue 18, for example a tumor or an inflammation, is shown, to which peptides 1 are increasingly attached. The distribution of 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: Via the blood circulation system 10, the peptide 1 is transported into the organs 12, 13, 14, 15, and 16 of the organism.

Phase III: The circulating peptide 1 binds specifically to the epitope 5 of the antigen 4 and accumulates on the diseased tissue 18 because this forms the antigen 4.

Phase IV: Unbound peptide 1 is rapidly metabolised and enzymatically degraded. The organism does not distinguish between its own peptides and the peptide 1, because it is made up of amino acids 2, 3, which correspond to the body's own molecules. The degradation products 17 of the peptide 1 and the amino acids 2, 3 accumulate predominantly in the kidney 16 from where they are excreted via the bladder and the ureter.

references

Colby DW, Garg P, Holden T, Chao G, Webster JM, Knife A, Ingram VN, Wittrup KD; Development of a human chain variable domain (V (L)) intracellular antibody specific for the amino terminus of huntingtin via yeast surface display; J Mol Biol. 2004 Sep 17; 342 (3): 901-12.

Divgi CR, Pandit Taskar N, Jungbluth AA, Reuter VE, Gonen M, Ruan S, Pierre C, Nagel Ά, Pryma DA, Humm J, Lareon SM, Old LJ, Russo P; Preoperative characterization of clear-cell renal carcinoma using iodine-124-labeled antibody chimeric G250 (124I-cG250) and PET in patients with renal masses: a phase I trial; Laneet Oncol. 2007 Apr; 8 {4): 304-10.

Faller A, Schünke M; The body of man; Thieme-Verlag; Of 2008.

Heap CJ, Wang Y, Pinheiro TJ, Reading SA, Jennings KR, Dimmock NJ; Analysis of a 17-amino acid residue, virus-neutralizing microantibody; J Gen Virol. 2005 Jun; 86 (Pt 6): 1791-800.

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. LIST OF REFERENCE NUMBERS

1 peptide

 2 amino acids

 3 N-tenninal amino acid

4 antigen / tumor antigen

5 epitope

 6 antibodies

 7 Paratop

 8 light polypeptide chain

9 heavy polypeptide chain

10 circulatory system

 11 (main) wheels

 12 lungs

 13 heart

 14 liver

 15 intestine

 16 kidney

 17 degradation products

 18 diseased tissue / tumor

Claims

claims Use of a peptide (1) for the production of an agent for the detection of an antigen (4), characterized in that the peptide (1)  a) an amino acid sequence of a paratope (7) of an antibody (6) which is directed against the antigen (4),  b) specifically binds to an epitope (5) of the antigen (4), and c) has an ¹¹ C carbon atom. Use according to claim 1, characterized in that the agent is a radiopharmaceutical. Use according to claim 1 or 2, characterized in that the antigen (4) is formed by a tumor (18). Use according to any one of the preceding claims, characterized in that the amino acid sequence is the amino acid sequence of a variable region of a polypeptide chain (8, 9) of the antibody (6). Use according to any one of the preceding claims, characterized in that 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 for the localization of a tumor (18) comprises a peptide (1) having an ¹¹ C carbon atom, characterized in that the peptide (1) has an amino acid sequence of a paratope (7) of an antibody (6) directed against the tumor (18). A radiopharmaceutical according to claim 6, characterized in that the amino acid sequence is the amino acid sequence of a variable region of a polypeptide chain (8,9) of the antibody (6). Radiopharmaceutical according to Claim 6 or 7, characterized in that 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 one of claims 6 to 8, characterized in that it is a positron emission tomography (PET) biomarker.