WO2012000866A1

WO2012000866A1 - 11c-labelled peptide for detecting a diseased tissue

Info

Publication number: WO2012000866A1
Application number: PCT/EP2011/060448
Authority: WO
Inventors: Oliver Lade; Jan Alexander Hiss; Hartmuth C. Kolb; Ursus KRÜGER; Gisbert Schneider; Arno Steckenborn
Original assignee: Siemens Aktiengesellschaft; Johann Wolfgang Goethe-Universität
Priority date: 2010-06-30
Filing date: 2011-06-22
Publication date: 2012-01-05
Also published as: DE102010026064A1

Abstract

The use of a peptide (1) to produce an agent for detecting a diseased tissue (18) which forms a human leukocyte antigen (HLA)-antigen complex (20) is described. In this case, the peptide (1) has an amino acid sequence of a complementary determining region (CDR) 3 loop (7) of a T-cell receptor (TCR) (6) directed against the HLA-antigen complex (20) and binds to an antigen (4) of the HLA-antigen complex (20). Furthermore, the peptide (1) also has a ¹¹C carbon atom. A radiopharmaceutical for locating a diseased tissue (18) which forms an HLA-antigen complex (20) is also described, which radiopharmaceutical comprises such a peptide (1).

Description

description

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 also endogenous substances are detected, which are ^¬ example, only formed in an infection by viruses or bacteria. These products include mainly components of the immune system, in particular antibodies that are formed from the Or ^¬ organism itself. Such in vitro studies may 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 Rönt ^¬ gen, ultrasound and magnetic resonance imaging are generally used. In particular, ectopic cell aggregates, such as tumors, or swellings of individual organs can be located with them. Shows an abnormal tissue but no significant morpho ^¬ logical abnormalities, or it is relatively small, it can be easily 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. These Object is achieved by the use of a peptide for the manufacture ^¬ development of an agent for detecting a pathological tissue, which forms a human leukocyte antigen (HLA) antigen complex. By the peptide, an amino acid sequence of a complementarity determining region (CDR) 3 loop of a T cell

Receptor (TCR), which is directed against the HLA antigen complex, and binds to the HLA antigen complex, the diseased tissue can be specifically detected. By having an ¹¹ C carbon atom, even a few cells of a diseased tissue can be localized by the radioactive signal.

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 peptide used in the present invention is selected to have an amino acid sequence of a CDR3 loop of a TCR directed against an HLA antigen complex of 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 whether they are of the body's own and foreign origin. If a cell presents foreign molecules, it is killed and removed by the immune system. The presentation of the fragments takes place in the form of HLA-antigen complexes. The term "HLA antigen complex" refers to a complex of an HLA transmembrane protein which is also "major histocompatible". bility complex "(MHC) is called, and an antigen. The term" antigen "denotes short-chain peptides or protein fragments, resulting in the degradation of their own and others pro ^¬ teinen in the cell and anchored by an HLA on the cell surface The extracellular part of the HLA-antigen complex is recognized and bound by T-cells of the immune system, based on the interactions between the antigen of the HLA-antigen complex and the T-cell TCR Two identical or different polypeptide chains (A, B) that bind together to form the HLA-antigen complex have been found so far: In total, four different human TCR polypeptide chains have been identified, designated alpha, beta, gamma and delta a highly variable region, the so-called complementarity determining region (CDR) 3 loop.) The amino acid sequence of the CDR3 loop is different in the TCR of each T cell and esp Takes the binding specificity of the receptor. Similar to an antibody, the TCR binds only a specific protein fragment (antigen) because the amino acids of the CDR3 loop interact specifically with the amino acids of the antigen. Depending on the nature of the polypeptide chain (alpha, beta, gamma, delta), the CDR3 loop may be about two to 21 amino acids in length. It usually comprises about 7 to 14 amino acids (Rock et al., 1994).

By having an amino acid sequence of a CDR3 loop of a TCR, the amino acid sequence of the peptide used in the present invention binds the same antigen as the TCR. To determine the Ami ^¬ acid sequence of a CDR3 loop are isolated T cells from the blood of a patient and grown clonally in culture. Since a T cell expresses only an individual TCR, the cells of a T cell clone all produce the same TCR. From the cells of a clone the DNA Se ^acid sequence of the TCR or the CDR3 loop is Polymerasenketten- reactions with specific primers amplified and sequenced. Subsequently, a peptide with this sequence is Herge ^¬ provides that has the same binding specificities as the TCR. Preferably, the peptide is chosen so that the bond between the peptide and the HLA antigen complex is a linear coefficient called. KD value of <100 nM, be ^¬ vorzugt of <10 nM, most preferably of 7, 5 nM on ^¬ points. With this peptide, the HLA-antigen complex can be detected on the surface of the diseased tissue.

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, viruses or bacteria infected cells hy pertrophes tissue, inflamed tissue and organs, hyperplasti ^¬ MOORISH and neoplastic tissue, such as ulcers, tumors and cancers. 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 so that they can be recognized by a TCR. By specifically binding the antigen of the HLA-antigen complex, the peptide allows a 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, which move away from each other at an angle of 180 °, ie exactly in the opposite direction. The photons can be detected and from this the position of the positron emission, or the ¹¹ C- Carbon atoms, to be calculated. , 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 method described in the patent applications DE 10 2009 035 648.7 and DE 10 2009 035 465.2, are suitable. Furthermore, the amount of peptides that is located at a certain point 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, 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 complex, with which it forms a stable compound that is difficult to access for enzymatic degradation. Free, unbound peptides, however rapidly metabolized and excreted from the Or ^¬ organism because they are degraded rapidly by endogenous enzymes. This results in a strong and specific signal at the position of the HLA-antigen complex, and the background signal is minimized.

In an advantageous embodiment of the invention, the agent is a radiopharmaceutical. The term "radiopharmaceuticals" records 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 radiation emitting 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 ethylenediaminetetraacetate (EDTA) bound to mono- or polysaccharides The nuclides are, depending on the nature of their radiation, by scintigraphy, Single Photon Emission Computed Tomography (SPECT) or positron emission tomography (PET) is detected. Because of their non-physiological constituents parts but conventional radiopharmaceuticals can Nebenwirkun ^¬ gen as anaphylactic or allergic reactions that cause the Kör ^¬ by a patient. The use of a peptide from endogenous amino acids reduces this risk significantly because neither the peptide itself, nor its degradation products are toxic. in addition, carbon, unlike Techne ^¬ consortium or xenon, egg n 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 can be used directly after the on ^¬ bring the ¹¹ C-labeled amino acid. ¹¹ C-carbon has a half-life of only about 20 Minu ^¬ th, so that the radiation dose must be chosen the higher, the more time is between the synthesis of the peptide and its ^¬ 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 between the processing of the ¹¹ 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 less expensive and thus 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 the half-life of a protein or peptide to Longer side ^¬ liked by also D-amino acids are used in addition to L-amino acids (Neundorf I et al., 2008). Thus, the pharmacological clearance, ie the time until the peptide from the Or- is excreted, 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 possibility of the pharmacological clearance of the peptide to stunning ^¬ influence, consists of individual amino acids of the peptide to be replaced by non-natural amino acids with similar chemical properties. The non-natural amino acids are metabolized more slowly because the body's own proteolytic enzymes are specially adapted to the degradation of natural amino acids. 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 comprising a peptide having an ¹¹ C carbon atom for the localization of a diseased tissue which forms an HLA antigen complex. The peptide has an amino acid sequence of a complementary determining region (CDR) 3 loop of a TCR directed against the HLA-antigen complex. As a result, the peptide binds specifically to the antigen of the HLA

Antigen complex, so that even a few cells of a sick ^¬ haften tissue, which forms this HLA antigen complex, can be specifically ^¬ fisch demonstrated. 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. They ^bind to the HLA antigen complex of diseased tissue and collect on its surface. This tissue can play as a center of inflammation to be infected cells or tumor by viruses or bacteria at ^¬. 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.

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 body. The PET also makes it possible to determine the amount of mar ^¬-labeled molecules quantitatively in a tissue.

Also disclosed is a method of localizing a diseased tissue in an organism, comprising the steps of a) providing a peptide, b) administering the peptide to the organism, c) detecting the peptide in the organism Organism using positron emission tomography (PET). In this case, the peptide has an amino acid sequence of a CDR3 loop of a T cell receptor (TCR), which is directed against the HLA antigen complex, and binds to an antigen of the HLA antigen complex. Furthermore, the peptide also has an ¹¹ C carbon atom.

With the peptide according to the invention an antigen of an HLA-antigen complex is tektiert de- inside an organism, and isolated, so that the distribution of an HLA antigen complex, observed in a patient's body ^¬ the can. In this way, for example, the size or extent of an infection or a tumor 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 are explained with reference to the attached schematic drawings.

FIG. 1A schematically shows a diseased tissue 18 on the surface of which a human leukocyte antigen (HLA) 5 with an antigen 4 is located. Together they form an HLA-antigen complex 20, to which in turn a T-cell receptor (TCR) 6 is attached. The TCR 6 consists of two different polypeptide chains (A, B) (8, 9), both of which have a highly variable region, a so-called CDR3 loop 7. The CDR3 loop 7 binds directly to the antigen 4.

The HLA 5 is formed by the cells of the diseased tissue 18 and presented on its surface. HLA 5 binds short peptides from proteins inside the cell descend and act as antigens 4. The TCR 6 recognizes the antigen 4 and binds the HLA-antigen complex 20. The specifi ^¬ specific binding affinity between the HLA-antigen complex 20 and the TCR 6 comes about due to chemical interactions between the antigen 4 and the CDR3 loop 7 , Its amino acid sequence determines the specificity of TCR 6, which recognizes and binds only one particular antigen 4.

FIG. 1B schematically shows peptide 1 bound to 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 amino acid sequence of the ¹¹ C-labeled peptide 1 corresponds to the amino acid sequence of the CDR3 loop 7 of the TCR 6, such that the peptide 1, the binding affinity of the CDR3 loop be sitting ^¬ 7 and specifically binds to the antigen. 4 Due to the binding specificity ser ^¬ the ¹¹ C-labeled peptide 1 for de- tektion 4 of the antigen may be used. The positrons released upon decay of the ¹¹ C carbon 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.

To analyze the amino acid sequence of the CDR3 loop 7 of the TCR 6, the DNA region of the CDR3 loop 7 of a TCR 6 is selectively amplified and sequenced. Subsequently, an ¹¹ C-labeled peptide 1, corresponding to the amino acid sequence of the CDR3 loop 7, is produced. Peptide 1 is administered to the patient in the form of a pharmaceutical composition binds to the antigen 4 and accumulates in the cells of the krankhaf ^¬ th tissue 18, such as a tumor. This accumulation is seen in positron emission tomography (PET). bar, so that the distribution of the antigen 4 or the position of the tumor 18 in the body of the patient can be determined. In this way, newly formed metastases carrying the HLA-antigen complex 20 are detected by means of PET.

FIG. 2 shows a representation of a peptide having the sequence SEQ ID NO: 1 by means of a chemical formula.

The peptide comprises 9 amino acids 2 of the following sequence: arginine-serine-glycine-tyrosine-asparagine-threonine-aspartate-lysine-leucine-isoleucine.

The N-terminal arginine 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 N-terminal arginine 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 completing the peptide synthesis with the ¹¹ C-labeled amino acid, peptide 1 can be used immediately after radioactive labeling.

The peptide of SEQ ID NO: 1 has the sequence of the CDR3 loop 7 of a TCR 6, which is formed by a T cell of a patient with a diffuse large-cell B-cell lymphoma (DLBCL) (Yin Q et al., 2010). The DLBCL is a Malig ^¬ ne lymph node enlargement, in addition to the normal lymphocytes especially many lymphoblasts are formed. On the basis of the morphology of the lymphoblast, a distinction is made between among others, centroblastic and immunoblastic lymphomas. DLBCL is one of the aggressive non-Hodgkin's lymphomas and occurs at a frequency of approximately 3-5 / 100,000 persons per year.

From the peripheral blood of the DLBCL patient T cells are isolated and expanded in culture. The T cells produce the TCR 6 directed against the HLA antigen complex 20 located on the DLBCL. The clone of a single T cell is used to determine the amino acid sequence of the CDR3

Loop 7 of the TCR 6 of this T cell to analyze. Subsequently ^¬ ßend is a ¹¹ C-labeled peptide 1, which comprises this sequence on ^¬ and specifically binds to the antigen 4 prepared. The peptide of SEQ ID NO: 1 locates antigen 4 or DLBCL.

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 circulatory system 10 comprises various schematically represented organs, such as lung 12, heart 13, liver 14, intestine 15 and kidney 16, and the main arteries 11, which connect these organs. 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, are attached to the increased peptides. 1 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 antigen 4 and accumulates on the diseased tissue 18 because it presents the HLA-antigen complex 20 with the antigen 4 on its surface.

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 :

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

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.

Rock EP, Sibbald PR, Davis MM, Chien YH; CDR3 length in antigen-specific immune receptors; J Exp Med. 1994 Jan 1;

179 (1): 323-8.

Yin Q, Tan H, Chen S, Yang L, Ye J, Li Y; Characterization of conserved CDR3 sequence of TCR alpha and beta-chain genes in peripheral blood T-cells from patients with diffuse large B-cell lymphoma; Hematology. 2010 Feb; 15 (1): 48-57.

Claims

claims

Use of a peptide (1) for the preparation of an agent for detecting a diseased tissue (18) which forms a human leukocyte antigen (HLA) antigen complex (20),

characterized,

that the peptide (1)

a) an amino acid sequence of a complementary determinant region (CDR) 3 loop (7) of a T cell receptor (TCR) (6) which is directed against the HLA antigen complex (20),

b) binds to an antigen (4) of the HLA-antigen complex (20), and

c) has an ¹¹ C carbon atom.

2. Use according to claim 1,

characterized,

that the agent is a radiopharmaceutical.

3. Use according to claim 1 or 2,

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).

4. Radiopharmaceutical for the localization of a diseased tissue (18), which forms an HLA-antigen complex (20), ^{comprising ¬} a peptide (1) with an ¹¹ C-carbon atom, characterized

in that the peptide (1) has an amino acid sequence of a CDR3 loop (7) of a TCR (6) directed against the HLA-antigen complex (20) and to the antigen (4) of the HLA-antigen complex ( 20) binds. Radiopharmaceutical according to claim 4,

characterized,

Radiopharmaceutical according to claim 4 or 5,

characterized,

that it is a positron emission tomography (PET) biomarker.