EP1684804A2 - Binding molecules for the extra-domain b of fibronectin, used for the detection of atherosclerotic plaques - Google Patents

Binding molecules for the extra-domain b of fibronectin, used for the detection of atherosclerotic plaques

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Publication number
EP1684804A2
EP1684804A2 EP04790493A EP04790493A EP1684804A2 EP 1684804 A2 EP1684804 A2 EP 1684804A2 EP 04790493 A EP04790493 A EP 04790493A EP 04790493 A EP04790493 A EP 04790493A EP 1684804 A2 EP1684804 A2 EP 1684804A2
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Prior art keywords
seq
xaa
use according
amino acids
amino acid
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EP04790493A
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German (de)
French (fr)
Inventor
Dieter Heldmann
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Philogen SpA
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Schering AG
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/10—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody
    • A61K51/1018—Antibodies or immunoglobulins; Fragments thereof, the carrier being an antibody, an immunoglobulin or a fragment thereof, e.g. a camelised human single domain antibody or the Fc fragment of an antibody against material from animals or humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)

Definitions

  • Binding molecules for the extra domain B of fibronectin for the detection of atherosclerotic plaques Binding molecules for the extra domain B of fibronectin for the detection of atherosclerotic plaques
  • the present invention relates to the use of binding molecules for the extra domain B (ED-B) of fibronectin, for example of labeled antibodies or antibody fragments against the ED-B domain, such as L19 derivatives, as diagnostic reagents for the detection of ED-B
  • ED-B extra domain B
  • L19 derivatives labeled antibodies or antibody fragments against the ED-B domain
  • Atherosclerosis is a change in blood vessels that develops over many years and initially goes undetected. The development of atherosclerosis runs through different stages. If the endothelial cell layer of the arterial wall or its non-adherent surface is damaged as a result of mechanical or chemical damage, the resulting change in normal blood flow promotes the attachment and aggregation of platelets, especially on the branches and branches of the arterial network, which leads to formation of blood clots, so-called thrombi, in the arterial walls.
  • Atherosclerosis develops quietly and secretly and causes no symptoms for a long time. Only when the vascular diameter is increasingly reduced due to the progressive formation of atherosclerotic plaques, the symptoms develop slowly but steadily. Since calcifications of the atherosclerotic plaques that have already occurred cannot be broken down and the elasticity cannot be restored to the resulting rigid artery walls, but the progression of the disease can be slowed down considerably when it is known, easy-to-perform atherosclerosis detection procedures with high sensitivity and specificity are of particular importance.
  • Contrast-enhanced angiography is an examination method for radiologically imaging blood vessels. Depending on which organ or which body region is to be depicted, a hollow needle or catheter is inserted into an artery, vein or tissue under local anesthesia. Then a contrast medium or marker is injected and the corresponding body region is X-rayed. Both arteries, veins and lymphatic drainage channels are described, which allows conclusions to be drawn about the type and extent of the disease. However, this method is significantly limited by the available contrast media and markers. These only allow a relatively unspecific representation of the room in which they are located, such as of the blood space, which makes the detection of atherosclerotic plaques considerably more difficult.
  • the multilayer spiral CT is an alternative to contrast-enhanced angiography.
  • it also offers the advantage of high-resolution contrast-enhanced CT angiography and thus enables the visualization of uncalcified plaques.
  • initial clinical studies already show clear limitations of the method, so that an unreflected clinical use cannot generally be recommended.
  • Doppler sonography is an ultrasound examination that uses the Doppler procedure and is used to diagnose heart diseases. Doppler sonography provides information about the direction and speed of blood flow, which can be used to detect narrowing of the arterial cavities. However, visualization of atherosclerotic plaque tissue is not possible with Doppler sonography.
  • Electron beam tomography is a method that allows a non-invasive determination of the extent of coronary atherosclerosis.
  • the progression of coronary atherosclerosis can also be assessed using electron beam tomography.
  • a disadvantage of the method is that it is very expensive and requires the purchase of an electron beam tomograph. It is therefore an object of the present invention to provide an alternative method for the detection of atherosclerotic plaques in arterial walls, which has a high sensitivity and specificity and enables simple, fast and inexpensive primary prevention.
  • This object is achieved according to the invention by using binding molecules against the ED-B of fibronectin for the detection of atherosclerotic processes, in particular for the detection of atherosclerotic plaques, including uncalcified and / or calcified plaques.
  • Binding molecules for the ED-B domain of fibronectin a sequence of 91 amino acids, which are inserted into the fibronectin molecule by alternative splicing (Castellani et al. (1994), Int. J. Cancer 59, 612-618), are already present in WO 97/45544, WO 01/62800 and WO 03/055917.
  • Preferred binding molecules are molecules which bind directly and specifically to the ED-B domain, such as antibodies against the ED-B domain or fragments of such antibodies, for example antibody fragments obtainable by proteolytic cleavage, for example Fab-, Fab'-, F (ab) 2 fragments etc. or recombinant antibody fragments, for example single-chain Fv fragments.
  • the ED-B binding molecules are preferably used as conjugates with labeling groups suitable for diagnostic applications.
  • a preferred embodiment of the invention relates to the use of the antibody L19 or fragments of this antibody (L19 derivatives), which are present as conjugates with labeling groups, for the production of a pharmaceutical composition for the detection of atherosclerotic plaques.
  • L19 is the scFv fragment (scFv: Single chain variable antibody fragment) of a monoclonal antibody against the extra domain B (ED-B) of fibronectin and has the following amino acid sequence (SEQ ID NO.1):
  • Linker GDGSSGGSGG ASTG (VL):
  • L19 has already been mentioned several times in the prior art. For example, Tarli et al. (Blood, Vol. 94, No. 1 (1999), pp. 192-198) the biodistribution of the highly affine human 125 l-labeled L19 in tumor-bearing mice with advanced angiogenesis in the area of the tumor tissue. Furthermore WO 01/62800 discloses the use of radioactively labeled conjugates, which comprise the scFv fragment L19, for the detection and treatment of angiogenesis. However, the use of labeled L19 derivatives for the detection of atherosclerotic plaques is neither disclosed nor suggested in the prior art.
  • the subject of the present invention therefore relates in particular to the use of a labeled L19 derivative comprising (aa) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR3 and / or LCDR3 or shown in Table 1 a variant of it, which is a deletion, Has insertion and / or substitution of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region, the antigen binding site performing the same function as that in SEQ ID NO. 1 native L19 shown,
  • (ab) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or a variant thereof, which deletion, insertion and / / or substitution of up to 3 amino acids in the HCDR1 region, up to 8 amino acids in the HCDR2 region, up to 5 amino acids in the HCDR3 region, up to 6 amino acids in the LCDR1 region, up to 4 Amino acids in the LCDR2 region and up to 6 amino acids in the LCDR3 region, wherein the antigen binding site has the same function as that in SEQ ID NO. 1 native L19 shown, or
  • (ac) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the one in SEQ ID NO. 1 shown sequence of the native L19 or a variation thereof, which has a deletion, insertion and / or substitution of up to 30 amino acids, the antigen binding site having the same function as that in SEQ ID NO. 1 native L19 shown, and optionally
  • the labeled L19 derivative comprises an N-terminal antigen binding site for the extra domain B (ED-B) of fibronectin selected from the antigen binding sites (aa), (ab) or (ac) and optionally a C-terminal Amino acid sequence selected from the amino acid sequences (ba), (bb), (bc) or (bd), the antigen binding site having the same function as that in SEQ ID NO. 1 native L19 shown.
  • ED-B extra domain B
  • the antigen binding sites (aa), (ab) and (ac) mediate a bond between the labeled L19 derivative and the atherosclerotic plaques, the complex of labeled L19 derivative and atherosclerotic plaque having a dissociation constant in the subnanomolar range (for example less than 10 "9 M).
  • the dissociation constant of the complex of labeled L19 derivative and atherosclerotic plaque is preferably in the same range as the dissociation constant of the complex of L19 derivative and the antigen ED-B fibronectin described in WO 99/58570.
  • the antigen binding sites for the extra domain B (ED-B) of fibronectin of the labeled L19 derivative (aa) and (ab) comprise the complementarity-determining regions HCDR3 and / or LCDR3 or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3.
  • the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined as follows:
  • HCDRx complementarity determining region x of the heavy antibody chain
  • LCDRx complementarity-determining region x of the light antibody chain.
  • CDR length length of the complementarity determining region.
  • a variant of the HCDR1 region comprises a deletion, insertion and / or substitution of up to 3 amino acids in the HCDR1 region, ie a deletion, insertion and / or substitution of 1, 2 or 3 amino acids with respect to the sequence shown in Table 1 (SEQ ID NO. 8).
  • a variant of the HCDR2 region comprises a deletion, insertion and / or substitution of up to 8 amino acids in the HCDR2 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7 or 8 amino acids related to the sequence shown in Table 1 (SEQ ID NO. 9).
  • a variant of the HCDR3 region comprises a deletion, insertion and / or substitution of up to 5 amino acids in the HCDR3 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4 or 5 amino acids with respect to the sequence shown in Table 1 (SEQ ID NO. 10).
  • a variant of the LCDR1 region comprises a deletion, insertion and / or substitution of up to 6 amino acids in the LCDR1 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4, 5 or 6 amino acids in Reference to the sequence shown in Table 1 (SEQ ID NO. 11).
  • a variant of the LCDR2 region comprises a deletion, insertion and / or substitution of up to 4 amino acids in the LCDR2 region, ie a deletion, insertion and / or substitution of 1, 2, 3 or 4 amino acids with respect to the Sequence shown in Table 1 (SEQ ID NO. 12).
  • a variant of the LCDR3 region comprises a deletion, insertion and / or substitution of up to 6 amino acids in the LCDR3 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4, 5 or 6 amino acids in relation to the sequence shown in Table 1 (SEQ ID NO. 13).
  • the antigen binding site for the extra domain B (ED-B) of fibronectin of the labeled L19 derivative (ac) comprises the one in SEQ ID NO. 1 shown sequence of the native L19 or a variation thereof, which has a deletion, insertion and / or substitution of up to 30 amino acids, i.e. a deletion, insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids with respect to those in SEQ ID NO. 1 sequence shown.
  • amino acid sequences (ba), (bb) and (bc) of the labeled L19 derivative include the sequences Xaa ⁇ -Xaa 2 -Xaa -Cys (SEQ ID NO. 2), Xaa ⁇ -Xaa 2 - Xaa 3 -Cys-Xaa 4 (SEQ ID NO. 3) and (His) favour(SEQ ID NO. 4 ).
  • amino acid sequence (ba) XaarXaa 2 -Xaa 3 -Cys is the sequence Gly-Gly-Gly-Cys (SEQ ID NO. 14) or Gly-Cys-Gly- Cys (SEQ ID NO.15).
  • the sequence Gly-Gly-Gly-Cys (SEQ ID NO. 14) is particularly preferred.
  • amino acid sequence (bb) XaarXaa 2 -Xaa 3 -Cys-Xaa is the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) or Gly -Cys-Gly-C s-Ala (SEQ ID NO. 17).
  • the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) is particularly preferred.
  • amino acid sequence (bc) (His) n (SEQ ID NO. 4) is the sequence (His) 6 with n equal to 6 (SEQ ID NO. 18).
  • the N-terminus of (aa), (ab) or (ac) is optionally connected via a peptide bond to the G-terminus of a linker amino acid sequence.
  • the linker amino acid sequence preferably has a length of up to 30 amino acids, preferably up to 25 amino acids and particularly preferably up to 22 amino acids.
  • the linker amino acid sequence that is shown in SEQ ID NO. 19 is the sequence shown.
  • labeled L19 derivatives include those in SEQ ID NO. 1 (native L19), SEQ ID NO. 20 (AP38), SEQ ID NO. 21 (AP39), SEQ ID NO. 22 (L19-GlyCysGlyCys), SEQ ID NO. 23 (L19-GlyCysGlyCysAla), SEQ ID NO. 24 (ZK225293), SEQ ID NO. 25 (ZK217691 / 217695), SEQ ID NO. 26 (ZK210917) and SEQ ID NO. 27 (ZK248219 / 248220) sequences shown.
  • the binding molecule for the ED-B domain is preferably in the form of a conjugate with a labeling substance. All marking substances suitable for diagnostic applications, in particular diagnostic applications in vivo, are suitable as marking substances, for example radioactive marking substances or for non-radioactive detection methods, eg marking substances suitable for magnetic resonance methods.
  • the binding molecule is preferably labeled with a radioisotope, for example a radioisotope of iodine (I), indium (In), technetium (Tc) and rhenium (Re).
  • a radioisotope for example a radioisotope of iodine (I), indium (In), technetium (Tc) and rhenium (Re).
  • the radioisotopes 125 l, 111 ln, 186 Re are particularly preferred.
  • an antibody fragment e.g. an L19 derivative in reduced form.
  • reduced form means that the fragment is in monomeric form and not in dimeric or multimeric form mediated by intermolecular disulfide bridges.
  • the reduced form of the antibody fragment is preferably obtained by adding a suitable reducing agent.
  • suitable reducing agents are well known in the art and include TCEP (tris (2-carboxyethyl) phosphine) and 1,4-dimercapto-2,3-butanediols.
  • the present invention provides that, in addition to the binding molecule, the pharmaceutical composition optionally contains physiologically compatible auxiliaries, carriers and / or diluents. Suitable auxiliaries, carriers and / or diluents are well known to those skilled in the field of pharmaceutical chemistry.
  • the atherosclerotic plaques are preferably detected in the Within the scope of the present invention by injecting the pharmaceutical composition comprising the ED-B binding molecule into a vein and / or artery of a patient to be examined and detecting the labeled ED-B binding molecule, if present, bound to the atherosclerotic plaque. If a radioisotope-labeled binding molecule is used, the detection can be carried out by scintigraphy.
  • the early detection of atherosclerotic plaques according to the present invention can prevent heart attacks and angina pectoris attacks, but also strokes, macular degeneration on the eye and / or thromboses.
  • L19 dehvates are prepared as described in WO 03/055917, the content of which is incorporated herein by reference.
  • Labeled L19 derivatives are prepared as described in WO 03/055917, the content of which is incorporated herein by reference.
  • Example 3 Investigation of the binding of different, labeled L19 derivatives to atherosclerotic vascular samples from WHHL rabbits in a special in vitro perfusion apparatus
  • This Perfusion apparatus contained vascular samples from the aorta of WHHL rabbits (Watanabe heritable hyperlipidemic rabbits).
  • WHHL rabbits develop atherosclerotic plaques due to a genetic defect in certain sections of the aorta.
  • Vascular samples from these atherosclerotic sections of the aorta were therefore used as a model for the disease atherosclerosis in humans.
  • Vessel samples from non-atherosclerotic sections of the aorta from the same rabbit were used as comparison controls.
  • the vascular samples were positioned in the vascular apparatus in such a way that the labeled L19 derivative to be examined could only bind to the luminal side of the aorta.
  • a solution of the labeled L19 derivative was perfused using a peristaltic pump at a rate of 1 ml / min. Perfusion was carried out over 20 min at room temperature. The volume of the perfusion circuit was 9 ml.
  • the labeled L19 derivative according to the invention was contained in the volume shown in Table 2 in this volume.
  • ZK225293 SEQ ID NO. 24
  • the evaluation factor for ZK225293 determined in the investigation was 4.5.
  • the result of this investigation shows the excellent potential of the labeled L19 derivative for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
  • ZK217691 / 217695 SEQ ID NO. 25
  • the evaluation factor for ZK2176691 / 217695 determined in the investigation was 8.7.
  • the result of this investigation shows the excellent potential of the labeled L19 Derivatives for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
  • ZK210917 SEQ ID NO. 26
  • the evaluation factor for ZK210917 determined in the investigation was 3.4.
  • the result of this investigation shows the excellent potential of the labeled L19 derivative for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
  • ZK217052 / 217053 SEQ ID NO. 21
  • the evaluation factor for ZK217052 / 217053 determined in the investigation was 4.8.
  • the result of this investigation shows the excellent potential of the labeled L19 derivative for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
  • Example 4 Investigation of the imaging of atherosclerotic plaques by means of the 99m Tc-labeled L19 derivative ZK248219 / 248220 in WHHL rabbits in vivo
  • the suitability of ZK248219 / 248220 was investigated in vivo on a WHHL rabbit. These WHHL rabbits develop atherosclerotic plaques due to a genetic defect in certain sections of the aorta and have therefore been used as a model for atherosclerosis in humans.
  • the test animal (3.4 kg body weight) was administered under anesthesia (Rompun / Ketavet (1: 2), 1 ml / kg body weight in) 41 MBq of the 99 ⁇ Tc-labeled L19 derivative ZK248219 / 248220 into the ear vein.
  • Whole body scans were taken with the ⁇ counter ( ⁇ camera Elscint SP4 HR) over a period of 5 hours. After 5 hours, the test animal was sacrificed and its aorta was autoradiographically examined to determine the precise distribution of the activity bound to the aorta.
  • the result of the imaging examination shows a clear representation of the aortic arch up to the point in time after injection.
  • the autoradiographic examination shows that the activity concentration in the aortic arch of the test animal is 12 times higher than in the PIaque-free, abdominal aortic areas.
  • the result of this investigation clearly shows the excellent potential of the labeled L19 derivative ZK248219 / 248220 for the diagnosis of atherosclerotic plaques.

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Abstract

The invention relates to the use of marked L19 derivatives for producing a pharmaceutical composition that is utilized for detecting atherosclerotic plaques.

Description

Bindemoleküle für die Extra-Domäne B von Fibronectin zur Detektion von atherosklerotischen Plaques Binding molecules for the extra domain B of fibronectin for the detection of atherosclerotic plaques
Beschreibung Die vorliegende Erfindung betrifft die Verwendung von Bindemolekülen für die Extra-Domäne B (ED-B) von Fibronectin, beispielsweise von markierten Antikörpern bzw. Antikörperfragmenten gegen die ED-B Domäne, wie etwa L19-Derivaten, als diagnostische Reagenzien zur Detektion vonThe present invention relates to the use of binding molecules for the extra domain B (ED-B) of fibronectin, for example of labeled antibodies or antibody fragments against the ED-B domain, such as L19 derivatives, as diagnostic reagents for the detection of
* atherosklerotischen Prozessen, insbesondere von atherosklerotischen Plaques. * atherosclerotic processes, especially atherosclerotic plaques.
Atherosklerose ist eine Veränderung der Blutgefäße, die über viele Jahre entsteht und zunächst unerkannt verläuft. Die Entwicklung einer Atherosklerose verläuft dabei über verschiedene Stufen. Kommt es zu einer Verletzung der endothelialen Zellschicht der Arterienwand oder deren nicht- haftender Oberfläche infolge mechanischer oder chemischer Verletzung, so fördert die daraus resultierende Veränderung des normalen Blutflusses die Anheftung und Aggregation von Blutplättchen, insbesondere an den Ästen und Abzweigungen des Arteriengeflechts, was zur Ausbildung von Blutgerinseln, sog. Thromben, in den Arterienwänden führen kann. Mit der Zeit führt die Anhäufung von Fettstreifen, eine Ansammlung von Schaumzellen, welche sich infolge der Thrombenbildung aus Monocyten des zellulären Abwehrsystems bilden, zu einer kontinuierlichen Zelleinwanderung, Cholesterinablagerung, Ausdehnung der glatten Muskulatur sowie Bildung von zusätzlichem Bindegewebe, wodurch es zu immer größeren Verletzungen kommt. Diese fortgeschrittenen Läsionen stellen schließlich die sogenannten atherosklerotischen Plaques dar, welche sich an der inneren Gefäßwand befinden, wo sie anschwellen und den Innenraum der Arterie einengen. Im weiteren Verlauf werden die atherosklerotischen Plaques dann bald mit einer dicken Schicht Bindegewebe überzogen. Mit der Zeit verkalken diese Plaques und es kommt zu weiteren Veränderungen, wie z.B. Rissen oder Blutungen, die zu einem teilweisen oder totalen Verschluss der Arterie führen können. Sobald kein Blut mehr fließen kann, sind die dahinter gelegenen Gewebe und Zellen von der Versorgung ausgeschlossen. Als Folge der Verengung können Herzinfarkte sowie Angina pectoris Anfälle, aber auch Schlaganfälle, Makuladegenerationen am Auge oder Thrombosen auftreten.Atherosclerosis is a change in blood vessels that develops over many years and initially goes undetected. The development of atherosclerosis runs through different stages. If the endothelial cell layer of the arterial wall or its non-adherent surface is damaged as a result of mechanical or chemical damage, the resulting change in normal blood flow promotes the attachment and aggregation of platelets, especially on the branches and branches of the arterial network, which leads to formation of blood clots, so-called thrombi, in the arterial walls. Over time, the accumulation of fat streaks, a collection of foam cells, which form as a result of thrombus formation from monocytes of the cellular defense system, leads to continuous cell immigration, cholesterol deposition, expansion of the smooth muscles and the formation of additional connective tissue, which leads to increasing injuries , These advanced lesions ultimately represent the so-called atherosclerotic plaques, which are located on the inner wall of the vessel, where they swell and constrict the interior of the artery. In the further course, the atherosclerotic plaques will soon be covered with a thick layer of connective tissue. Over time, these plaques calcify and further changes, such as cracks or bleeding, occur partial or total occlusion of the artery. As soon as blood can no longer flow, the tissues and cells behind it are excluded from the supply. As a result of the narrowing, heart attacks and angina pectoris attacks, but also strokes, macular degeneration on the eye or thrombosis can occur.
Die Atherosklerose entwickelt sich still und heimlich und verursacht lange keine Symptome. Erst wenn der Gefäßdurchmesser durch die fortschreitende Bildung der atherosklerotischen Plaques zunehmend reduziert wird, entstehen die Symptome langsam, aber stetig. Da bereits eingetretene Verkalkungen der atherosklerotischen Plaques nicht abgebaut werden können und den daraus resultierenden starren Arterienwänden die Elastizität nicht zurückgegeben werden kann, das Fortschreiten der Krankheit jedoch bei deren Kenntnis deutlich verlangsamt werden kann, sind einfach durchführbare Atherosklerosedetektionsverfahren mit hoher Sensitivität und Spezifität von besonderer Bedeutung.Atherosclerosis develops quietly and secretly and causes no symptoms for a long time. Only when the vascular diameter is increasingly reduced due to the progressive formation of atherosclerotic plaques, the symptoms develop slowly but steadily. Since calcifications of the atherosclerotic plaques that have already occurred cannot be broken down and the elasticity cannot be restored to the resulting rigid artery walls, but the progression of the disease can be slowed down considerably when it is known, easy-to-perform atherosclerosis detection procedures with high sensitivity and specificity are of particular importance.
Bislang wurden verschiedene Untersuchungsmethoden zur Diagnostik der Atherosklerose entwickelt, darunter kontrastmittelverstärkte Angiographie, Mehrschicht Spiral-CT, Doppler-Sonographie, Magnetresonanztomographie und Elektronenstrahltomographie.To date, various examination methods for the diagnosis of atherosclerosis have been developed, including contrast-enhanced angiography, multilayer spiral CT, Doppler sonography, magnetic resonance imaging and electron beam tomography.
Die kontrastmittelverstärkte Angiographie ist eine Untersuchungsmethode, um Blutgefäße radiologisch darzustellen. Je nachdem, welches Organ oder welche Körperregion dargestellt werden soll, wird in örtlicher Betäubung eine Hohlnadel oder ein Katheter in eine Arterie, Vene oder in das Gewebe eingeführt. Danach wird ein Kontrastmittel oder Marker eingespritzt und die entsprechende Körperregion wird geröntgt. Dabei werden sowohl Arterien, Venen als auch Lymphabflussbahnen beschrieben, wodurch Rückschlüsse auf die Art und die Ausdehnung der Erkrankung möglich sind. Eine wesentliche Limitation erfährt diese Methode jedoch durch die verfügbaren Kontrastmittel und Marker. Diese ermöglichen lediglich eine relativ unspezifische Darstellung des Raumes in dem sie sich befinden, wie z.B. des Blutraumes, wodurch die Detektion der atherosklerotischen Plaques erheblich erschwert wird.Contrast-enhanced angiography is an examination method for radiologically imaging blood vessels. Depending on which organ or which body region is to be depicted, a hollow needle or catheter is inserted into an artery, vein or tissue under local anesthesia. Then a contrast medium or marker is injected and the corresponding body region is X-rayed. Both arteries, veins and lymphatic drainage channels are described, which allows conclusions to be drawn about the type and extent of the disease. However, this method is significantly limited by the available contrast media and markers. These only allow a relatively unspecific representation of the room in which they are located, such as of the blood space, which makes the detection of atherosclerotic plaques considerably more difficult.
Die Mehrschicht Spiral-CT stellt eine Alternative zur kontrastmittelverstärkten Angiographie dar. Neben der validen Dokumentation verkalkter atherosklerotischer Plaques bietet sie zusätzlich den Vorteil hochauflösender kontrastmittelverstärkter CT-Angiographie und ermöglicht damit auch die Visualisierung unverkalkter Plaques. Erste klinische Studien zeigen jedoch bereits deutliche Limitationen der Methode, so dass ein unreflektierter klinischer Einsatz generell nicht empfohlen werden kann.The multilayer spiral CT is an alternative to contrast-enhanced angiography. In addition to the valid documentation of calcified atherosclerotic plaques, it also offers the advantage of high-resolution contrast-enhanced CT angiography and thus enables the visualization of uncalcified plaques. However, initial clinical studies already show clear limitations of the method, so that an unreflected clinical use cannot generally be recommended.
Die Doppler-Sonographie ist eine Ultraschall-Untersuchung, bei der das Doppler-Verfahren zum Einsatz kommt und dient der Diagnose von Herzerkrankungen. Durch die Doppler-Sonographie werden Informationen über Richtung und Geschwindigkeit des Blutflusses erhalten, wodurch Einengungen der Hohlräume von Arterien festgestellt werden können. Eine Visualisierung von atherosklerotischem Plaquegewebe ist hingegen mit der Doppler-Sonographie nicht möglich.Doppler sonography is an ultrasound examination that uses the Doppler procedure and is used to diagnose heart diseases. Doppler sonography provides information about the direction and speed of blood flow, which can be used to detect narrowing of the arterial cavities. However, visualization of atherosclerotic plaque tissue is not possible with Doppler sonography.
Die Magnetresonanztomographie ermöglicht durch Verwendung verschiedener Pulssequenzen eine Visualisierung von atherosklerotischem Plaquegewebe sowie eine Gewebecharakterisierung der einzelnen Plaquekomponenten. Der Einsatz dieser Methode zur Primärprävention bedarf jedoch noch erheblicher Weiterentwicklung.By using different pulse sequences, magnetic resonance tomography enables visualization of atherosclerotic plaque tissue as well as tissue characterization of the individual plaque components. However, the use of this method for primary prevention still requires considerable further development.
Mit der Elektronenstrahltomographie steht ein Verfahren zur Verfügung, das eine nicht-invasive Bestimmung des Ausmaßes der koronaren Atherosklerose erlaubt. Mittels Elektronenstrahltomographie kann somit auch die Progression der koronaren Atherosklerose beurteilt werden. Ein Nachteil der Methode ist jedoch, dass sie sehr teuer ist und der Anschaffung eines Elektronenstrahltomographen bedarf. Aufgabe der vorliegenden Erfindung ist es demzufolge ein alternatives Verfahren zur Detektion von atherosklerotischen Plaques in Arterienwänden bereitzustellen, das eine hohe Sensitivität und Spezifität aufweist und eine einfache, schnelle und kostengünstige Primärprävention möglich macht.Electron beam tomography is a method that allows a non-invasive determination of the extent of coronary atherosclerosis. The progression of coronary atherosclerosis can also be assessed using electron beam tomography. A disadvantage of the method, however, is that it is very expensive and requires the purchase of an electron beam tomograph. It is therefore an object of the present invention to provide an alternative method for the detection of atherosclerotic plaques in arterial walls, which has a high sensitivity and specificity and enables simple, fast and inexpensive primary prevention.
Diese Aufgabe wird erfindungsgemäß gelöst durch Verwendung von Bindemolekülen gegen die ED-B von Fibronectin zur Detektion von atherosklerotischen Prozessen, insbesondere zur Detektion von atherosklerotischen Plaques, einschließlich unverkalkter oder/und verkalkter Plaques.This object is achieved according to the invention by using binding molecules against the ED-B of fibronectin for the detection of atherosclerotic processes, in particular for the detection of atherosclerotic plaques, including uncalcified and / or calcified plaques.
Bindemoleküle für die ED-B Domäne von Fibronectin, einer Sequenz von 91 Aminosäuren, die durch alternatives Spleißen in das Fibronectin-Molekül insertiert wird (Castellani et al. (1994), Int. J. Cancer 59, 612-618), sind bereits in WO 97/45544, WO 01/62800 und WO 03/055917 beschrieben. Bevorzugte Bindemoleküle sind Moleküle, die direkt und spezifisch an die ED-B Domäne binden, wie etwa Antikörper gegen die ED-B Domäne oder Fragmente solcher Antikörper, beispielsweise durch proteolytische Spaltung erhältliche Antikörperfragmente, z.B. Fab-, Fab'-, F(ab) 2-Fragmente etc. oder rekombinante Antikörperfragmente, z.B. einzelkettige Fv-Fragmente. Die ED-B-Bindemoleküle werden vorzugsweise als Konjugate mit für diagnostische Anwendungen geeigneten Markierungsgruppen eingesetzt.Binding molecules for the ED-B domain of fibronectin, a sequence of 91 amino acids, which are inserted into the fibronectin molecule by alternative splicing (Castellani et al. (1994), Int. J. Cancer 59, 612-618), are already present in WO 97/45544, WO 01/62800 and WO 03/055917. Preferred binding molecules are molecules which bind directly and specifically to the ED-B domain, such as antibodies against the ED-B domain or fragments of such antibodies, for example antibody fragments obtainable by proteolytic cleavage, for example Fab-, Fab'-, F (ab) 2 fragments etc. or recombinant antibody fragments, for example single-chain Fv fragments. The ED-B binding molecules are preferably used as conjugates with labeling groups suitable for diagnostic applications.
Eine bevorzugte Ausführungsform der Erfindung betrifft die Verwendung des Antikörpers L19 bzw. Fragmenten dieses Antikörpers (L19-Derivate), die als Konjugate mit Markierungsgruppen vorliegen, zur Herstellung einer pharmazeutischen Zusammensetzung zur Detektion von atherosklerotischen Plaques. Überraschenderweise konnte mit Hilfe der der vorliegenden Erfindung zugrunde liegenden Studien festgestellt werden, dass atherosklerotische Plaques hochspezifisch und mit hoher Sensitivität durch Bindung markierter L19-Derivate diagnostiziert werden können.A preferred embodiment of the invention relates to the use of the antibody L19 or fragments of this antibody (L19 derivatives), which are present as conjugates with labeling groups, for the production of a pharmaceutical composition for the detection of atherosclerotic plaques. Surprisingly, with the help of the studies on which the present invention is based, it was found that atherosclerotic plaques can be diagnosed in a highly specific manner and with high sensitivity by binding labeled L19 derivatives.
L19 ist das scFv-Fragment (scFv: Single chain variable antibody fragment) eines monoklonalen Antikörpers gegen die Extra-Domäne B (ED-B) von Fibronectin und weist folgende Aminosäuresequenz (SEQ ID NO.1) auf:L19 is the scFv fragment (scFv: Single chain variable antibody fragment) of a monoclonal antibody against the extra domain B (ED-B) of fibronectin and has the following amino acid sequence (SEQ ID NO.1):
(VH): EVQLLESGGG LVQPGGSLRL SCAASGFTFS(VH): EVQLLESGGG LVQPGGSLRL SCAASGFTFS
SFSMSWVRQA PGKGLEWVSS ISGSSGTTYYSFSMSWVRQA PGKGLEWVSS ISGSSGTTYY
ADSVKGRFTI SRDNSKNTLY LQMNSLRAEDADSVKGRFTI SRDNSKNTLY LQMNSLRAED
TAVYYCAKPF PYFDYWGQGT LVTVSSTAVYYCAKPF PYFDYWGQGT LVTVSS
(Linker): GDGSSGGSGG ASTG (VL):(Linker): GDGSSGGSGG ASTG (VL):
EIVLTQSPGT LSLSPGERAT LSCRASQSVSEIVLTQSPGT LSLSPGERAT LSCRASQSVS
SSFLAWYQQK PGQAPRLLIY YASSRATGIPSSFLAWYQQK PGQAPRLLIY YASSRATGIP
DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QTGRIPPTFG QGTKVEIKDRFSGSGSGT DFTLTISRLE PEDFAVYYCQ QTGRIPPTFG QGTKVEIK
L19 ist bereits verschiedentlich im Stand der Technik erwähnt. So beschreiben Tarli et al. (Blood, Vol. 94, No. 1 (1999), S. 192-198) die Bioverteilung des hoch affinen humanen 125l-markierten L19 in Tumor- tragenden Mäusen mit fortgeschrittener Angiogenese im Bereich des Tumorgewebes. Des Weiteren offenbart WO 01/62800 die Verwendung von radioaktiv markierten Konjugaten, welche das scFv-Fragment L19 umfassen, zum Nachweis und zur Behandlung von Angiogenese. Die Verwendung von markierten L19-Derivaten zur Detektion von atherosklerotischen Plaques ist hingegen im Stand der Technik weder offenbart noch nahegelegt.L19 has already been mentioned several times in the prior art. For example, Tarli et al. (Blood, Vol. 94, No. 1 (1999), pp. 192-198) the biodistribution of the highly affine human 125 l-labeled L19 in tumor-bearing mice with advanced angiogenesis in the area of the tumor tissue. Furthermore WO 01/62800 discloses the use of radioactively labeled conjugates, which comprise the scFv fragment L19, for the detection and treatment of angiogenesis. However, the use of labeled L19 derivatives for the detection of atherosclerotic plaques is neither disclosed nor suggested in the prior art.
Der Gegenstand der vorliegenden Erfindung betrifft daher insbesondere die Verwendung eines markierten L19-Derivats, umfassend (aa) mindestens eine Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin umfassend die in Tabelle 1 gezeigten komplementaritätsbestimmenden Regionen HCDR3 und/oder LCDR3 oder eine Variante davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 5 Aminosäuren in der HCDR3-Region und von bis zu 6 Aminosäuren in der LCDR3-Region aufweist, wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19,The subject of the present invention therefore relates in particular to the use of a labeled L19 derivative comprising (aa) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR3 and / or LCDR3 or shown in Table 1 a variant of it, which is a deletion, Has insertion and / or substitution of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region, the antigen binding site performing the same function as that in SEQ ID NO. 1 native L19 shown,
(ab) mindestens eine Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin umfassend die in Tabelle 1 gezeigten komplementaritätsbestimmenden Regionen HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 und LCDR3 oder eine Variante davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 3 Aminosäuren in der HCDR1 -Region, von bis zu 8 Aminosäuren in der HCDR2-Region, von bis zu 5 Aminosäuren in der HCDR3-Region, von bis zu 6 Aminosäuren in der LCDR1 -Region, von bis zu 4 Aminosäuren in der LCDR2- Region und von bis zu 6 Aminosäuren in der LCDR3-Region aufweist, wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19, oder(ab) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or a variant thereof, which deletion, insertion and / / or substitution of up to 3 amino acids in the HCDR1 region, up to 8 amino acids in the HCDR2 region, up to 5 amino acids in the HCDR3 region, up to 6 amino acids in the LCDR1 region, up to 4 Amino acids in the LCDR2 region and up to 6 amino acids in the LCDR3 region, wherein the antigen binding site has the same function as that in SEQ ID NO. 1 native L19 shown, or
(ac) mindestens eine Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin umfassend die in SEQ ID NO. 1 gezeigte Sequenz des nativen L19 oder eine Variation davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 30 Aminosäuren aufweist, wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19, und gegebenenfalls(ac) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the one in SEQ ID NO. 1 shown sequence of the native L19 or a variation thereof, which has a deletion, insertion and / or substitution of up to 30 amino acids, the antigen binding site having the same function as that in SEQ ID NO. 1 native L19 shown, and optionally
(ba) eine Aminosäuresequenz Xaaι-Xaa2-Xaa3-Cys (SEQ ID NO. 2), wobei Xaai, Xaa2 und Xaa3 unabhängig voneinander jede natürlich vorkommende Aminosäure darstellen,(ba) an amino acid sequence Xaaι-Xaa 2 -Xaa 3 -Cys (SEQ ID NO. 2), where Xaai, Xaa 2 and Xaa 3 independently represent each naturally occurring amino acid,
(bb) eine Aminosäuresequenz XaarXaa2-Xaa3-Cys-Xaa (SEQ ID NO. 3), wobei Xaai, Xaa2, Xaa3 und Xaa unabhängig voneinander jede natürlich vorkommende Aminosäure darstellen,(bb) an amino acid sequence XaarXaa 2 -Xaa 3 -Cys-Xaa (SEQ ID NO. 3), where Xaai, Xaa 2 , Xaa 3 and Xaa independently represent each naturally occurring amino acid,
(bc) eine Aminosäuresequenz (His)n (SEQ ID NO. 4), wobei n eine ganze Zahl von 4 bis 6 ist, oder (bd) eine Aminosäuresequenz umfassend die in SEQ ID NO. 5, SEQ ID NO. 6 oder SEQ ID NO. 7 gezeigte Sequenz, wobei der C-Terminus von (aa), (ab) oder (ac) gegebenenfalls über eine Peptidbindung an den N-Terminus von (ba), (bb), (bc) oder (bd) gebunden ist, zur Herstellung einer pharmazeutischen Zusammensetzung zur Detektion von atherosklerotischen Plaques.(bc) an amino acid sequence (His) n (SEQ ID NO. 4), where n is a is an integer from 4 to 6, or (bd) an amino acid sequence comprising the one in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7 sequence shown, wherein the C-terminus of (aa), (ab) or (ac) is optionally bound via a peptide bond to the N-terminus of (ba), (bb), (bc) or (bd) for Preparation of a pharmaceutical composition for the detection of atherosclerotic plaques.
Das markierte L19-Derivat umfasst im Rahmen der vorliegenden Erfindung eine N-terminale Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin ausgewählt aus den Antigenbindungsstellen (aa), (ab) oder (ac) und gegebenenfalls eine C-terminale Aminosäuresequenz ausgewählt aus den Aminosäuresequenzen (ba), (bb), (bc) oder (bd), wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19. Gemäß vorliegender Erfindung bedeutet dies, dass die Antigenbindungsstellen (aa), (ab) und (ac) des markierten L19-Derivats eine im wesentlichen gleiche Bindungskonstante an atherosklerotische Plaques aufweisen, wie das in SEQ ID NO. 1 gezeigte native scFv-Fragment L19. Insbesondere vermitteln die Antigenbindungsstellen (aa), (ab) und (ac) eine Bindung zwischen dem markierten L19-Derivat und den atherosklerotischen Plaques, wobei der Komplex aus markiertem L19-Derivat und atherosklerotischem Plaque eine Dissoziationskonstante im subnanomolaren Bereich aufweist (z.B. geringer als 10 "9 M). Vorzugsweise liegt die Dissoziationskonstante des Komplexes aus markiertem L19-Derivat und atherosklerotischem Plaque im selben Bereich wie die in WO 99/58570 beschriebene Dissoziationskonstante des Komplexes aus L19-Derivat und dem Antigen ED-B Fibronectin.In the context of the present invention, the labeled L19 derivative comprises an N-terminal antigen binding site for the extra domain B (ED-B) of fibronectin selected from the antigen binding sites (aa), (ab) or (ac) and optionally a C-terminal Amino acid sequence selected from the amino acid sequences (ba), (bb), (bc) or (bd), the antigen binding site having the same function as that in SEQ ID NO. 1 native L19 shown. According to the present invention, this means that the antigen binding sites (aa), (ab) and (ac) of the labeled L19 derivative have an essentially identical binding constant to atherosclerotic plaques as that in SEQ ID NO. 1 native scFv fragment L19 shown. In particular, the antigen binding sites (aa), (ab) and (ac) mediate a bond between the labeled L19 derivative and the atherosclerotic plaques, the complex of labeled L19 derivative and atherosclerotic plaque having a dissociation constant in the subnanomolar range (for example less than 10 "9 M). The dissociation constant of the complex of labeled L19 derivative and atherosclerotic plaque is preferably in the same range as the dissociation constant of the complex of L19 derivative and the antigen ED-B fibronectin described in WO 99/58570.
Die Antigenbindungsstellen für die Extra-Domäne B (ED-B) von Fibronectin des markierten L19-Derivats (aa) bzw. (ab) umfassen gemäß vorliegender Erfindung die in Tabelle 1 gezeigten komplementaritätsbestimmenden Regionen HCDR3 und/oder LCDR3 bzw. HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 und LCDR3. Im Rahmen der vorliegenden Erfindung sind die komplementaritätsbestimmenden Regionen HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 und LCDR3 wie folgt definiert:According to the present invention, the antigen binding sites for the extra domain B (ED-B) of fibronectin of the labeled L19 derivative (aa) and (ab) comprise the complementarity-determining regions HCDR3 and / or LCDR3 or HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3. In the context of the present invention, the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 are defined as follows:
Tabelle 1Table 1
(1)HCDRx: komplementaritätsbestimmende Region x der schweren Antikörperkette; LCDRx: komplementaritätsbestimmende Region x der leichten Antikörperkette. (2)CDR-Länge: Länge der komplementaritätsbestimmenden Region. (1) HCDRx: complementarity determining region x of the heavy antibody chain; LCDRx: complementarity-determining region x of the light antibody chain. (2) CDR length: length of the complementarity determining region.
Neben den in Tabelle 1 definierten komplementaritätsbestimmenden Regionen, können die Antigenbindungsstellen für die Extra-Domäne B (ED- B) von Fibronectin des markierten L19-Derivats (aa) bzw. (ab) auch Varianten dieser Regionen umfassen. Erfindungsgemäß umfasst eine Variante der HCDR1 -Region eine Deletion, Insertion und/oder Substitution von bis zu 3 Aminosäuren in der HCDR1 -Region, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2 oder 3 Aminosäuren in Bezug auf die in Tabelle 1 gezeigte Sequenz (SEQ ID NO. 8). Eine Variante der HCDR2- Region umfasst eine Deletion, Insertion und/oder Substitution von bis zu 8 Aminosäuren in der HCDR2-Region, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2, 3, 4, 5, 6, 7 oder 8 Aminosäuren in Bezug auf die in Tabelle 1 gezeigte Sequenz (SEQ ID NO. 9). Zudem umfasst eine Variante der HCDR3-Region eine Deletion, Insertion und/oder Substitution von bis zu 5 Aminosäuren in der HCDR3-Region, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2, 3, 4 oder 5 Aminosäuren in Bezug auf die in Tabelle 1 gezeigte Sequenz (SEQ ID NO. 10). Eine Variante der LCDR1 -Region umfasst hingegen eine Deletion, Insertion und/oder Substitution von bis zu 6 Aminosäuren in der LCDR1 -Region, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2, 3, 4, 5 oder 6 Aminosäuren in Bezug auf die in Tabelle 1 gezeigte Sequenz (SEQ ID NO. 11 ). Des Weiteren umfasst eine Variante der LCDR2-Region eine Deletion, Insertion und/oder Substitution von bis zu 4 Aminosäuren in der LCDR2-Region, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2, 3 oder 4 Aminosäuren in Bezug auf die in Tabelle 1 gezeigte Sequenz (SEQ ID NO. 12). Eine Variante der LCDR3-Region umfasst eine Deletion, Insertion und/oder Substitution von bis zu 6 Aminosäuren in der LCDR3-Region, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2, 3, 4, 5 oder 6 Aminosäuren in Bezug auf die in Tabelle 1 gezeigte Sequenz (SEQ ID NO. 13).In addition to the complementarity-determining regions defined in Table 1, the antigen binding sites for the extra domain B (ED-B) of fibronectin of the labeled L19 derivative (aa) or (ab) can also comprise variants of these regions. According to the invention, a variant of the HCDR1 region comprises a deletion, insertion and / or substitution of up to 3 amino acids in the HCDR1 region, ie a deletion, insertion and / or substitution of 1, 2 or 3 amino acids with respect to the sequence shown in Table 1 (SEQ ID NO. 8). A variant of the HCDR2 region comprises a deletion, insertion and / or substitution of up to 8 amino acids in the HCDR2 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7 or 8 amino acids related to the sequence shown in Table 1 (SEQ ID NO. 9). In addition, a variant of the HCDR3 region comprises a deletion, insertion and / or substitution of up to 5 amino acids in the HCDR3 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4 or 5 amino acids with respect to the sequence shown in Table 1 (SEQ ID NO. 10). A variant of the LCDR1 region, however, comprises a deletion, insertion and / or substitution of up to 6 amino acids in the LCDR1 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4, 5 or 6 amino acids in Reference to the sequence shown in Table 1 (SEQ ID NO. 11). Furthermore, a variant of the LCDR2 region comprises a deletion, insertion and / or substitution of up to 4 amino acids in the LCDR2 region, ie a deletion, insertion and / or substitution of 1, 2, 3 or 4 amino acids with respect to the Sequence shown in Table 1 (SEQ ID NO. 12). A variant of the LCDR3 region comprises a deletion, insertion and / or substitution of up to 6 amino acids in the LCDR3 region, ie a deletion, insertion and / or substitution of 1, 2, 3, 4, 5 or 6 amino acids in relation to the sequence shown in Table 1 (SEQ ID NO. 13).
Die Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin des markierten L19-Derivats (ac) umfasst gemäß vorliegender Erfindung die in SEQ ID NO. 1 gezeigte Sequenz des nativen L19 oder eine Variation davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 30 Aminosäuren aufweist, d.h. eine Deletion, Insertion und/oder Substitution von 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 oder 30 Aminosäuren in Bezug auf die in SEQ ID NO. 1 gezeigte Sequenz.The antigen binding site for the extra domain B (ED-B) of fibronectin of the labeled L19 derivative (ac) according to the present invention comprises the one in SEQ ID NO. 1 shown sequence of the native L19 or a variation thereof, which has a deletion, insertion and / or substitution of up to 30 amino acids, i.e. a deletion, insertion and / or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids with respect to those in SEQ ID NO. 1 sequence shown.
Die Aminosäuresequenzen (ba), (bb) bzw. (bc) des markierten L19-Derivats umfassen die Sequenzen Xaaι-Xaa2-Xaa -Cys (SEQ ID NO. 2), Xaaι-Xaa2- Xaa3-Cys-Xaa4 (SEQ ID NO. 3) bzw. (His)„ (SEQ ID NO. 4).The amino acid sequences (ba), (bb) and (bc) of the labeled L19 derivative include the sequences Xaaι-Xaa 2 -Xaa -Cys (SEQ ID NO. 2), Xaaι-Xaa 2 - Xaa 3 -Cys-Xaa 4 (SEQ ID NO. 3) and (His) „(SEQ ID NO. 4 ).
In einer bevorzugten Ausführungsform der vorliegenden Erfindung ist die Aminosäuresequenz (ba) XaarXaa2-Xaa3-Cys (SEQ ID NO. 2) die Sequenz Gly-Gly-Gly-Cys (SEQ ID NO. 14) oder Gly-Cys-Gly-Cys (SEQ ID NO. 15). Besonders bevorzugt ist die Sequenz Gly-Gly-Gly-Cys (SEQ ID NO. 14).In a preferred embodiment of the present invention, the amino acid sequence (ba) XaarXaa 2 -Xaa 3 -Cys (SEQ ID NO. 2) is the sequence Gly-Gly-Gly-Cys (SEQ ID NO. 14) or Gly-Cys-Gly- Cys (SEQ ID NO.15). The sequence Gly-Gly-Gly-Cys (SEQ ID NO. 14) is particularly preferred.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung ist die Aminosäuresequenz (bb) XaarXaa2-Xaa3-Cys-Xaa (SEQ ID NO. 3) die Sequenz Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) oder Gly-Cys-Gly-C s- Ala (SEQ ID NO. 17). Besonders bevorzugt ist die Sequenz Gly-Gly-Gly- Cys-Ala (SEQ ID NO. 16).In a further preferred embodiment of the present invention, the amino acid sequence (bb) XaarXaa 2 -Xaa 3 -Cys-Xaa (SEQ ID NO. 3) is the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) or Gly -Cys-Gly-C s-Ala (SEQ ID NO. 17). The sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) is particularly preferred.
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung ist die Aminosäuresequenz (bc) (His)n (SEQ ID NO. 4) die Sequenz (His)6 mit n gleich 6 (SEQ ID NO. 18).In a further preferred embodiment of the present invention, the amino acid sequence (bc) (His) n (SEQ ID NO. 4) is the sequence (His) 6 with n equal to 6 (SEQ ID NO. 18).
In einer weiteren bevorzugten Ausführungsform der vorliegenden Erfindung ist der N-Terminus von (aa), (ab) oder (ac) gegebenenfalls über eine Peptidbindung mit dem G-Terminus einer Linkeraminosäuresequenz verbunden ist. Vorzugsweise weist die Linkeraminosäuresequenz eine Länge von bis zu 30 Aminosäuren, bevorzugt bis zu 25 Aminosäuren und besonders bevorzugt bis zu 22 Aminosäuren auf. Besonders bevorzugt ist die Linkeraminosäuresequenz die in SEQ ID NO. 19 gezeigte Sequenz ist.In a further preferred embodiment of the present invention, the N-terminus of (aa), (ab) or (ac) is optionally connected via a peptide bond to the G-terminus of a linker amino acid sequence. The linker amino acid sequence preferably has a length of up to 30 amino acids, preferably up to 25 amino acids and particularly preferably up to 22 amino acids. The linker amino acid sequence that is shown in SEQ ID NO. 19 is the sequence shown.
Gemäß vorliegender Erfindung umfassen besonders bevorzugte markierte L19-Derivate die in SEQ ID NO. 1 (natives L19), SEQ ID NO. 20 (AP38), SEQ ID NO. 21 (AP39), SEQ ID NO. 22 (L19-GlyCysGlyCys), SEQ ID NO. 23 (L19-GlyCysGlyCysAla), SEQ ID NO. 24 (ZK225293), SEQ ID NO. 25 (ZK217691/217695), SEQ ID NO. 26 (ZK210917) und SEQ ID NO. 27 (ZK248219/248220) gezeigten Sequenzen. Das Bindemolekül für die ED-B-Domäne liegt vorzugsweise in Form eines Konjugats mit einer Markierungssubstanz vor. Als Markierungssubstanzen sind sämtliche für diagnostische Anwendungen, insbesondere diagnostische Anwendungen in vivo geeigneten Markierungsubstanzen, geeignet, beispielsweise radioaktive Markierungssubstanzen oder für nicht radioaktive Nachweismethoden, z.B. für Magnetresonanzverfahren geeignete Markierungssubεtanzen.According to the present invention, particularly preferred labeled L19 derivatives include those in SEQ ID NO. 1 (native L19), SEQ ID NO. 20 (AP38), SEQ ID NO. 21 (AP39), SEQ ID NO. 22 (L19-GlyCysGlyCys), SEQ ID NO. 23 (L19-GlyCysGlyCysAla), SEQ ID NO. 24 (ZK225293), SEQ ID NO. 25 (ZK217691 / 217695), SEQ ID NO. 26 (ZK210917) and SEQ ID NO. 27 (ZK248219 / 248220) sequences shown. The binding molecule for the ED-B domain is preferably in the form of a conjugate with a labeling substance. All marking substances suitable for diagnostic applications, in particular diagnostic applications in vivo, are suitable as marking substances, for example radioactive marking substances or for non-radioactive detection methods, eg marking substances suitable for magnetic resonance methods.
Verfahren zur Einführung von Markierungssubstanzen in Polypeptide, Peptide und insbesondere scFv-Fragmente sind im Stand der Technik gut bekannt. Vorzugsweise ist das Bindemolekül mit einem Radioisotop, z.B. einem Radioisotop von lod (I), Indium (In), Technetium (Tc) und Rhenium (Re) markiert. Besonders bevorzugt sind die Radioisotope 125l, 111ln, 186Re,Methods for introducing labeling substances into polypeptides, peptides and in particular scFv fragments are well known in the art. The binding molecule is preferably labeled with a radioisotope, for example a radioisotope of iodine (I), indium (In), technetium (Tc) and rhenium (Re). The radioisotopes 125 l, 111 ln, 186 Re, are particularly preferred.
188Rθ] 94mTc odθr 99mT 188 Rθ] 94m Tc or 99m T
In einer bevorzugten Ausführungsform der vorliegenden Erfindung wird ein Antikörperfragment, z.B. ein L19-Derivat in reduzierter Form, verwendet. Im Rahmen der vorliegenden Erfindung bedeutet der Begriff „reduzierte Form", dass das Fragment in monomerer Form und nicht etwa in durch intermolekulare Disulfidbrücken vermittelter dimerer oder multimerer Form vorliegt. Vorzugsweise wird die reduzierte Form des Antikörperfragments durch Zugabe eines geeigneten Reduktionsmittels erhalten. Geeignete Reduktionsmittel sind im Stand der Technik gut bekannt und umfassen TCEP (Tris(2-carboxyethyl)phosphin) und 1 ,4-Dimercapto-2,3-butandiole.In a preferred embodiment of the present invention an antibody fragment, e.g. an L19 derivative in reduced form. In the context of the present invention, the term “reduced form” means that the fragment is in monomeric form and not in dimeric or multimeric form mediated by intermolecular disulfide bridges. The reduced form of the antibody fragment is preferably obtained by adding a suitable reducing agent. Suitable reducing agents are well known in the art and include TCEP (tris (2-carboxyethyl) phosphine) and 1,4-dimercapto-2,3-butanediols.
Des Weiteren sieht die vorliegende Erfindung vor, dass die pharmazeutische Zusammensetzung zusätzlich zu dem Bindemolekül gegebenenfalls physiologisch verträgliche Hilfsmittel, Träger und/oder Verdünnungsmittel enthält. Geeignete Hilfsmittel, Träger und/oder Verdünnungsmittel sind dem Fachmann im Bereich der pharmazeutischen Chemie bestens bekannt.Furthermore, the present invention provides that, in addition to the binding molecule, the pharmaceutical composition optionally contains physiologically compatible auxiliaries, carriers and / or diluents. Suitable auxiliaries, carriers and / or diluents are well known to those skilled in the field of pharmaceutical chemistry.
Vorzugsweise erfolgt die Detektion der atherosklerotischen Plaques im Rahmen der vorliegenden Erfindung durch Injizieren der pharmazeutischen Zusammensetzung, welche das ED-B-Bindemolekül umfasst, in eine Vene und/oder Arterie eines zu untersuchenden Patienten und Detektieren des an die atherosklerotischen Plaques - falls vorhanden - gebundenen markierten ED-B-Bindemoleküls. Wird ein Radioisotop-markiertes Bindemolekül verwendet, so kann die Detektion durch Szintigraphie erfolgen. Durch die frühzeitige Detektion von atherosklerotischen Plaques gemäß vorliegender Erfindung kann Herzinfarkten sowie Angina pectoris Anfällen, aber auch Schlaganfällen, Makuladegenerationen am Auge oder/und Thrombosen vorgebeugt werden.The atherosclerotic plaques are preferably detected in the Within the scope of the present invention by injecting the pharmaceutical composition comprising the ED-B binding molecule into a vein and / or artery of a patient to be examined and detecting the labeled ED-B binding molecule, if present, bound to the atherosclerotic plaque. If a radioisotope-labeled binding molecule is used, the detection can be carried out by scintigraphy. The early detection of atherosclerotic plaques according to the present invention can prevent heart attacks and angina pectoris attacks, but also strokes, macular degeneration on the eye and / or thromboses.
Des Weiteren wird die vorliegende Erfindung durch Figur 1 und die nachfolgenden Beispiele näher erläutert.Furthermore, the present invention is illustrated by Figure 1 and the following examples.
BeispieleExamples
Beispiel 1: Herstellung von L19-DerivatenExample 1: Preparation of L19 derivatives
Die Herstellung von L19-Dehvaten erfolgt wie in WO 03/055917 beschrieben, auf deren Inhalt hierin Bezug genommen wird.L19 dehvates are prepared as described in WO 03/055917, the content of which is incorporated herein by reference.
Beispiel 2: Markierung der L19-Derivate mit Hilfe von RadioisotopenExample 2: Labeling of the L19 derivatives using radioisotopes
Die Herstellung markierter L19-Derivate erfolgt wie in WO 03/055917 beschrieben, auf deren Inhalt hierin Bezug genommen wird.Labeled L19 derivatives are prepared as described in WO 03/055917, the content of which is incorporated herein by reference.
Beispiel 3: Untersuchung der Bindung verschiedener, markierter L19- Derivate an atherosklerotische Gefäßproben von WHHL-Kaninchen in einer speziellen in vitro PerfusionsapparaturExample 3: Investigation of the binding of different, labeled L19 derivatives to atherosclerotic vascular samples from WHHL rabbits in a special in vitro perfusion apparatus
Zur Untersuchung der Eignung verschiedener, markierter L19-Derivate wurde eine in vitro Perfusionsapparatur (Ussing-Kammer) verwendet. Diese Perfusionsapparatur enthielt Gefäßproben aus der Aorta von WHHL- Kaninchen (Watanabe heritable hyperlipidemic rabbits).An in vitro perfusion apparatus (Ussing chamber) was used to investigate the suitability of various labeled L19 derivatives. This Perfusion apparatus contained vascular samples from the aorta of WHHL rabbits (Watanabe heritable hyperlipidemic rabbits).
Diese WHHL-Kaninchen entwickeln aufgrund eines genetischen Defekts in bestimmten Abschnitten der Aorta atherosklerotische Plaques. Gefäßproben aus diesen atherosklerotischen Abschnitten der Aorta wurden deshalb als Modell für die Krankheit Atherosklerose beim Menschen eingesetzt. Als Vergleichskontrolle wurden jeweils aus dem gleichen Kaninchen Gefäßproben aus nicht atherosklerotischen Abschnitten der Aorta verwendet.These WHHL rabbits develop atherosclerotic plaques due to a genetic defect in certain sections of the aorta. Vascular samples from these atherosclerotic sections of the aorta were therefore used as a model for the disease atherosclerosis in humans. Vessel samples from non-atherosclerotic sections of the aorta from the same rabbit were used as comparison controls.
Die Gefäßproben wurden in der Gefäßapparatur derart positioniert, dass das jeweilige zu untersuchende markierte L19-Derivat nur an der luminalen Seite der Aorta binden konnte. Eine Lösung des markierten L19-Derivats wurde dabei mit Hilfe einer peristaltischen Pumpe mit einer Geschwindigkeit von 1 ml/min perfundiert. Die Perfusion wurde über 20 min bei Raumtemperatur durchgeführt. Das Volumen des Perfusionskreislaufes betrug 9 ml. In diesem Volumen war das markierte erfindungsgemäße L19-Derivat in der in Tabelle 2 angegebenen Menge enthalten.The vascular samples were positioned in the vascular apparatus in such a way that the labeled L19 derivative to be examined could only bind to the luminal side of the aorta. A solution of the labeled L19 derivative was perfused using a peristaltic pump at a rate of 1 ml / min. Perfusion was carried out over 20 min at room temperature. The volume of the perfusion circuit was 9 ml. The labeled L19 derivative according to the invention was contained in the volume shown in Table 2 in this volume.
Nach abgeschlossener Perfusion wurde die an die atherosklerotischen Plaques der Aorta gebundene Menge des zu untersuchenden markiertenAfter perfusion was completed, the amount bound to the atherosclerotic plaques of the aorta was examined
L19-Derivat.es mit Hilfe eines γ-Counters (γ-Kamera Elscint SP4 HR) bestimmt. Aus dem Verhältnis der Menge des gebundenen markierten L19- Derivates an die atherosklerotischen und nicht-atherosklerotischen Abschnitte der Aorta ergibt sich der Bewertungsfaktor des jeweiligen markierten L19-Derivates. Tabelle 2L19- Derivat.es determined with the help of a γ-counter (γ-camera Elscint SP4 HR). The ratio of the amount of the labeled L19 derivative bound to the atherosclerotic and non-atherosclerotic sections of the aorta results in the evaluation factor of the respective labeled L19 derivative. Table 2
Beispiel 3.1 : Untersuchung des 125l-markierten L19-Derivates ZK225293Example 3.1: Examination of the 125 l-labeled L19 derivative ZK225293
Die Untersuchung der Eignung von ZK225293 (SEQ ID NO. 24) wurde wie in Beispiel 3 beschrieben durchgeführt. Der in der Untersuchung ermittelte Bewertungsfaktor für ZK225293 betrug 4,5. Das Ergebnis dieser Untersuchung zeigt das hervorragende Potential des markierten L19- Derivates zum Nachweis atherosklerotischer Plaques und damit zur Diagnose von Atherosklerose in Arterien.The suitability of ZK225293 (SEQ ID NO. 24) was examined as described in Example 3. The evaluation factor for ZK225293 determined in the investigation was 4.5. The result of this investigation shows the excellent potential of the labeled L19 derivative for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
Beispiel 3.2: Untersuchung des 25l-markierten L19-Derivates ZK212667Example 3.2: Examination of the 25 l-labeled L19 derivative ZK212667
Die Untersuchung der Eignung von ZK212667 (L19; SEQ ID NO. 1) wurde wie in Beispiel 3 beschrieben durchgeführt. Der in der Untersuchung ermittelte Bewertungsfaktor für ZK212667 betrug 2,8. Das Ergebnis dieserThe suitability of ZK212667 (L19; SEQ ID NO. 1) was tested as described in Example 3. The evaluation factor for ZK212667 determined in the investigation was 2.8. The result of this
Untersuchung zeigt das hervorragende Potential des markierten L19-Investigation shows the excellent potential of the labeled L19
Derivates zum Nachweis atherosklerotischer Plaques und damit zurDerivatives for the detection of atherosclerotic plaques and thus for
Diagnose von Atherosklerose in Arterien.Diagnosis of atherosclerosis in arteries.
Beispiel 3.3: Untersuchung des 111ln-markierten L19-DerivatesExample 3.3: Examination of the 111 ln-labeled L19 derivative
ZK217691/217695ZK217691 / 217695
Die Untersuchung der Eignung von ZK217691/217695 (SEQ ID NO. 25) wurde wie in Beispiel 3 beschrieben durchgeführt. Der in der Untersuchung ermittelte Bewertungsfaktor für ZK2176691/217695 betrug 8,7. Das Ergebnis dieser Untersuchung zeigt das hervorragende Potential des markierten L19- Derivates zum Nachweis atherosklerotischer Plaques und damit zur Diagnose von Atherosklerose in Arterien.The suitability of ZK217691 / 217695 (SEQ ID NO. 25) was tested as described in Example 3. The evaluation factor for ZK2176691 / 217695 determined in the investigation was 8.7. The result of this investigation shows the excellent potential of the labeled L19 Derivatives for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
Beispiel 3.4: Untersuchung des 111In-markierten L19-Derivat.es ZK210917Example 3.4: Examination of the 111 In-labeled L19 derivative ZK210917
Die Untersuchung der Eignung von ZK210917 (SEQ ID NO. 26) wurde wie in Beispiel 3 beschrieben durchgeführt. Der in der Untersuchung ermittelte Bewertungsfaktor für ZK210917 betrug 3,4. Das Ergebnis dieser Untersuchung zeigt das hervorragende Potential des markierten L19- Derivates zum Nachweis atherosklerotischer Plaques und damit zur Diagnose von Atherosklerose in Arterien.The suitability of ZK210917 (SEQ ID NO. 26) was examined as described in Example 3. The evaluation factor for ZK210917 determined in the investigation was 3.4. The result of this investigation shows the excellent potential of the labeled L19 derivative for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
Beispiel 3.5: Untersuchung des 99mTc-markierten L19-Derivates ZK217052/217053Example 3.5: Examination of the 99m Tc-labeled L19 derivative ZK217052 / 217053
Die Untersuchung der Eignung von ZK217052/217053 (SEQ ID NO. 21) wurde wie in Beispiel 3 beschrieben durchgeführt. Der in der Untersuchung ermittelte Bewertungsfaktor für ZK217052/217053 betrug 4,8. Das Ergebnis dieser Untersuchung zeigt das hervorragende Potential des markierten L19- Derivates zum Nachweis atherosklerotischer Plaques und damit zur Diagnose von Atherosklerose in Arterien.The suitability of ZK217052 / 217053 (SEQ ID NO. 21) was examined as described in Example 3. The evaluation factor for ZK217052 / 217053 determined in the investigation was 4.8. The result of this investigation shows the excellent potential of the labeled L19 derivative for the detection of atherosclerotic plaques and thus for the diagnosis of atherosclerosis in arteries.
Beispiel 4: Untersuchung der Bildgebung von atherosklerotischen Plaques mithilf e des 99mTc-markierten L19-Derivates ZK248219/248220 an WHHL-Kaninchen in vivoExample 4: Investigation of the imaging of atherosclerotic plaques by means of the 99m Tc-labeled L19 derivative ZK248219 / 248220 in WHHL rabbits in vivo
Die Untersuchung der Eignung von ZK248219/248220 (SEQ ID NO. 27) wurde in vivo an einem WHHL-Kaninchen durchgeführt. Diese WHHL- Kaninchen entwickeln aufgrund eines genetischen Defekts in bestimmten Abschnitten der Aorta atherosklerotische Plaques und wurden daher als Modell für die Krankheit Atherosklerose beim Menschen eingesetzt. Dem Versuchstier (3,4 kg Körpergewicht) wurde in Narkose (Rompun/Ketavet (1 :2), 1 ml/kg Körpergewicht i.m.) 41 MBq des 99πTc- markierten L19-Derivates ZK248219/248220 in die Ohrrandvene appliziert. Über einen Zeitraum von 5 h wurden mit dem γ-Counter (γ-Kamera Elscint SP4 HR) Ganzkörperszintigramme aufgenommen. Nach 5 h wurde das Versuchstier getötet und seine Aorta autoradiographisch untersucht, um die genaue Verteilung der an der Aorta gebundenen Aktivität zu bestimmen.The suitability of ZK248219 / 248220 (SEQ ID NO. 27) was investigated in vivo on a WHHL rabbit. These WHHL rabbits develop atherosclerotic plaques due to a genetic defect in certain sections of the aorta and have therefore been used as a model for atherosclerosis in humans. The test animal (3.4 kg body weight) was administered under anesthesia (Rompun / Ketavet (1: 2), 1 ml / kg body weight in) 41 MBq of the 99π Tc-labeled L19 derivative ZK248219 / 248220 into the ear vein. Whole body scans were taken with the γ counter (γ camera Elscint SP4 HR) over a period of 5 hours. After 5 hours, the test animal was sacrificed and its aorta was autoradiographically examined to determine the precise distribution of the activity bound to the aorta.
Das Ergebnis der Bildgebungs-Untersuchung zeigt bis zum Zeitpunkt post injectionem eine deutliche Darstellung des Aortabogens. Die autoradiographische Untersuchung belegt, dass im Aortabogen des Versuchstieres eine um den Faktor 12 höhere Aktivitätskonzentration als in den PIaque-freien, abdominalen Aortabereichen vorliegt. Das Ergebnis dieser Untersuchung zeigt also ganz deutlich das hervorragende Potential des markierten L19-Derivates ZK248219/248220 zur Diagnose atherosklerotischer Plaques. The result of the imaging examination shows a clear representation of the aortic arch up to the point in time after injection. The autoradiographic examination shows that the activity concentration in the aortic arch of the test animal is 12 times higher than in the PIaque-free, abdominal aortic areas. The result of this investigation clearly shows the excellent potential of the labeled L19 derivative ZK248219 / 248220 for the diagnosis of atherosclerotic plaques.

Claims

Ansprüche Expectations
1. Verwendung von Bindemolekülen gegen die Extra-Domäne B von Fibronectin zur Herstellung einer pharmazeutischen Zusammensetzung zur Detektion von atherosklerotischen Plaques.1. Use of binding molecules against the extra domain B of fibronectin for the production of a pharmaceutical composition for the detection of atherosclerotic plaques.
2. Verwendung nach Anspruch 1 , dadurch gekennzeichnet, dass die Bindemoleküle ausgewählt werden aus Antikörpern und Fragmenten davon.2. Use according to claim 1, characterized in that the binding molecules are selected from antibodies and fragments thereof.
3. Verwendung nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die Bindemoleküle eine Markierungsgruppe tragen.3. Use according to claim 1 or 2, characterized in that the binding molecules carry a labeling group.
4. Verwendung nach einem der Ansprüche 1 bis 3, dass die Bindemolekülen ausgewählt werden aus 19-Derivaten, umfassend (aa) mindestens eine Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin umfassend die in Tabelle 1 gezeigten komplementaritätsbestimmenden Regionen HCDR3 und/oder LCDR3 oder eine Variante davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 5 Aminosäuren in der HCDR3-Region und von bis zu 6 Aminosäuren in der LCDR3- Region aufweist, wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19, (ab) mindestens eine Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin umfassend die in Tabelle 1 gezeigten komplementaritätsbestimmenden Regionen HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 und LCDR3 oder eine Variante davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 3 Aminosäuren in der HCDR1 -Region, von bis zu 8 Aminosäuren in der HCDR2-Region, von bis zu 5 Aminosäuren in der _HCDR3-Region, von bis zu 6 Aminosäuren in der LCDR1 -Region, von bis zu 4 Aminosäuren in der LCDR2- Region und von bis zu 6 Aminosäuren in der LCDR3-Region aufweist, wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19, oder (ac) mindestens eine Antigenbindungsstelle für die Extra-Domäne B (ED-B) von Fibronectin umfassend die in SEQ ID NO. 1 gezeigte Sequenz des nativen L19 oder eine Variation davon, welche eine Deletion, Insertion und/oder Substitution von bis zu 30 Aminosäuren aufweist, wobei die Antigenbindungsstelle dieselbe Funktion aufweist, wie das in SEQ ID NO. 1 gezeigte native L19, und gegebenenfalls4. Use according to one of claims 1 to 3, that the binding molecules are selected from 19 derivatives, comprising (aa) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR3 shown in Table 1 and / or LCDR3 or a variant thereof which has a deletion, insertion and / or substitution of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region, the antigen binding site having the same function as that in SEQ ID NO. 1 shown native L19, (ab) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 or a variant shown in Table 1 Of which, a deletion, insertion and / or substitution of up to 3 amino acids in the HCDR1 region, of up to 8 amino acids in the HCDR2 region, of up to 5 amino acids in the _HCDR3 region, of up to 6 amino acids in the LCDR1 region, of up to 4 amino acids in the LCDR2 region and of up to 6 amino acids in the LCDR3 region, the antigen binding site performing the same function as that in SEQ ID NO. 1 shown native L19, or (ac) at least one antigen binding site for the extra domain B (ED-B) of fibronectin comprising the in SEQ ID NO. 1 shown sequence of the native L19 or a variation thereof, which has a deletion, insertion and / or substitution of up to 30 amino acids, the antigen binding site having the same function as that in SEQ ID NO. 1 native L19 shown, and optionally
(ba) eine Aminosäuresequenz Xaaι-Xaa2-Xaa -Cys (SEQ ID NO. 2), wobei Xaai, Xaa2 und Xaa3 unabhängig voneinander jede natürlich vorkommende Aminosäure darstellen, (bb) eine Aminosäuresequenz Xaaι-Xaa2-Xaa3-Cys-Xaa4 (SEQ ID NO. 3), wobei Xaa-i, Xaa2, Xaa3 und Xaa unabhängig voneinander jede natürlich vorkommende Aminosäure darstellen,(ba) an amino acid sequence Xaaι-Xaa 2 -Xaa -Cys (SEQ ID NO. 2), where Xaai, Xaa 2 and Xaa 3 independently represent each naturally occurring amino acid, (bb) an amino acid sequence Xaaι-Xaa 2 -Xaa 3 - Cys-Xaa 4 (SEQ ID NO. 3), where Xaa-i, Xaa 2 , Xaa 3 and Xaa independently represent each naturally occurring amino acid,
(bc) eine Aminosäuresequenz (His)n (SEQ ID NO. 4), wobei n eine ganze Zahl von 4 bis 6 ist, oder(bc) an amino acid sequence (His) n (SEQ ID NO. 4), where n is an integer from 4 to 6, or
(bd) eine Aminosäuresequenz umfassend die in SEQ ID NO. 5, SEQ ID NO. 6 oder SEQ ID NO. 7 gezeigte Sequenz, wobei der C-Terminus von (aa), (ab) oder (ac) gegebenenfalls über eine Peptidbindung an den N-Terminus von (ba), (bb), (bc) oder (bd) gebunden ist. (bd) an amino acid sequence comprising the one in SEQ ID NO. 5, SEQ ID NO. 6 or SEQ ID NO. 7 sequence shown, the C-terminus of (aa), (ab) or (ac) optionally being linked via a peptide bond to the N-terminus of (ba), (bb), (bc) or (bd).
5. Verwendung nach Anspruch 4, dadurch gekennzeichnet, dass die Aminosäuresequenz Xaaι-Xaa2-Xaa3-Cys die Sequenz Gly- Gly-Gly-Cys (SEQ ID NO. 14) oder Gly-Cys-Gly-Cys (SEQ ID NO. 15) ist.5. Use according to claim 4, characterized in that the amino acid sequence Xaaι-Xaa 2 -Xaa 3 -Cys the sequence Gly-Gly-Gly-Cys (SEQ ID NO. 14) or Gly-Cys-Gly-Cys (SEQ ID NO 15) is.
6. Verwendung nach Anspruch 4 oder 5, dadurch gekennzeichnet, dass die Aminosäuresequenz Xaaι-Xaa2-Xaa3-Cys-Xaa die Sequenz Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) oder Gly-Cys-Gly-Cys-Ala (SEQ ID NO. 17) ist.6. Use according to claim 4 or 5, characterized in that the amino acid sequence Xaaι-Xaa 2 -Xaa 3 -Cys-Xaa the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO. 16) or Gly-Cys-Gly -Cys-Ala (SEQ ID NO.17).
7. Verwendung nach einem der Ansprüche 4 bis 6, dadurch gekennzeichnet, dass n in der Aminosäuresequenz (His)n 6 (SEQ ID NO. 18) ist.7. Use according to one of claims 4 to 6, characterized in that n in the amino acid sequence (His) n is 6 (SEQ ID NO. 18).
8. Verwendung nach einem der Ansprüche 4 bis 7, dadurch gekennzeichnet, dass der N-Terminus von (aa), (ab) oder (ac) gegebenenfalls über eine Peptidbindung mit dem C-Terminus einer Linkeraminosäuresequenz verbunden ist.8. Use according to one of claims 4 to 7, characterized in that the N-terminus of (aa), (ab) or (ac) is optionally connected via a peptide bond to the C-terminus of a linker amino acid sequence.
9. Verwendung nach Anspruch 8, dadurch gekennzeichnet, dass die Linkeraminosäuresequenz eine Länge von bis zu 30 Aminosäuren aufweist.9. Use according to claim 8, characterized in that the linker amino acid sequence has a length of up to 30 amino acids.
10. Verwendung nach Anspruch 8 oder 9, dadurch gekennzeichnet, dass die Unkeraminosäuresequenz die in SEQ ID NO. 19 gezeigte Sequenz ist. 10. Use according to claim 8 or 9, characterized in that the unkeramino acid sequence that in SEQ ID NO. 19 is the sequence shown.
11. Verwendung nach einem der Ansprüche 4 bis 10, dadurch gekennzeichnet, dass das markierte L19-Derivat die in SEQ ID NO. 1 , SEQ ID NO. 20, SEQ ID NO. 21 , SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ I D NO. 25, SEQ ID NO. 26 oder SEQ ID NO. 27 gezeigte Sequenz umfasst.11. Use according to one of claims 4 to 10, characterized in that the labeled L19 derivative which in SEQ ID NO. 1, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26 or SEQ ID NO. 27 includes the sequence shown.
12. Verwendung nach einem der Ansprüche 1 bis 11 , dadurch gekennzeichnet, dass das Bindemolekül mit einem Radioisotop markiert ist.12. Use according to one of claims 1 to 11, characterized in that the binding molecule is labeled with a radioisotope.
13. Verwendung nach Anspruch 12, dadurch gekennzeichnet, dass das Radioisotop ausgewählt ist aus Radioisotopen von lod (I), Indium (In), Technetium (Tc) und Rhenium (Re).13. Use according to claim 12, characterized in that the radioisotope is selected from radioisotopes of iodine (I), indium (In), technetium (Tc) and rhenium (Re).
14. Verwendung nach Anspruch 12 oder 13, dadurch gekennzeichnet, dass das Radioisotop 125l, 111ln, 86Re, 188Re, 94mTc oder 99πTc ist.14. Use according to claim 12 or 13, characterized in that the radioisotope is 125 l, 111 ln, 86 Re, 188 Re, 94m Tc or 99π Tc.
15. Verwendung nach einem der Ansprüche 1 bis 14, dadurch gekennzeichnet, dass die Bindemoleküle aus Antikörperfragmenten, insbesondere L19-Derivaten in reduzierter Form, ausgewählt werden.15. Use according to one of claims 1 to 14, characterized in that the binding molecules are selected from antibody fragments, in particular L19 derivatives in reduced form.
16. Verwendung nach einem der Ansprüche 1 bis 15, dadurch gekennzeichnet, dass die pharmazeutische Zusammensetzung zusätzlich physiologisch verträgliche Hilfsmittel, Träger und/oder Verdünnungsmittel enthält. 16. Use according to one of claims 1 to 15, characterized in that the pharmaceutical composition additionally contains physiologically compatible auxiliaries, carriers and / or diluents.
17. Verwendung nach einem der Ansprüche 1 bis 16, dadurch gekennzeichnet, dass die Zusammensetzung zur Injektion in eine Vene und/oder Arterie eines Patienten vorgesehen ist.17. Use according to one of claims 1 to 16, characterized in that the composition is intended for injection into a vein and / or artery of a patient.
18. Verwendung nach einem der Ansprüche 1 bis 17, dadurch gekennzeichnet, dass die Zusammensetzung ein zur Detektion geeignetes radioaktiv markiertes Bindemolekül enthält.18. Use according to one of claims 1 to 17, characterized in that the composition contains a radioactive labeled binding molecule suitable for detection.
19. Verwendung nach einem der Ansprüche 1 bis 18 zur Prävention von Herzinfarkten sowie Angina pectoris Anfällen, aber auch Schlaganfällen, Makuladegenerationen am Auge oder Thrombosen.19. Use according to one of claims 1 to 18 for the prevention of heart attacks and angina pectoris attacks, but also strokes, macular degeneration on the eye or thrombosis.
20. Verfahren zur Detektion von atherosklerotischen Plaques, umfassend das Verabreichen eines Bindemoleküls gegen die Extra-Domäne B von Fibronectin in einer diagnostisch ausreichenden Menge an einen zu untersuchenden Patienten, insbesondere einen humanen Patienten, und Bestimmen der Lokalisation des Bindemoleküls in den Blutgefäßen des Patienten. 20. A method for the detection of atherosclerotic plaques, comprising administering a binding molecule against the extra domain B of fibronectin in a diagnostically sufficient amount to a patient to be examined, in particular a human patient, and determining the location of the binding molecule in the patient's blood vessels.
EP04790493A 2003-10-17 2004-10-15 Binding molecules for the extra-domain b of fibronectin, used for the detection of atherosclerotic plaques Ceased EP1684804A2 (en)

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WO2001062800A1 (en) * 2000-02-24 2001-08-30 Eidgenössische Technische Hochschule Zürich Antibody specific for the ed-b domain of fibronectin, conjugates comprising said antibody, and their use for the detection and treatment of angiogenesis
US7785591B2 (en) 2004-10-14 2010-08-31 Morphosys Ag Identification and characterization of function-blocking anti-ED-B-fibronectin antibodies
WO2008046510A1 (en) * 2006-10-16 2008-04-24 Bayer Healthcare Ag Fn1 as a biomarker, therapeutic and diagnostic target
EP1917980A1 (en) * 2006-10-31 2008-05-07 Bayer Schering Pharma Aktiengesellschaft Use of a fusion protein between a cytokine and an antibody targeting the ED-B fibronectin domain for the treatment of atherosclerosis
WO2011110490A1 (en) 2010-03-09 2011-09-15 Bayer Pharma Aktiengesellschaft Process for the production of radioactively labelled scfv antibody fragments, kits and compositions

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058570A2 (en) * 1998-05-11 1999-11-18 Eidgenössische Technische Hochschule Zürich Antibodies to the ed-b domain of fibronectin, conjugates containing them and use therefor for diagnosis and therapy of tumors and diseases associated with angiogenesis

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5576195A (en) * 1985-11-01 1996-11-19 Xoma Corporation Vectors with pectate lyase signal sequence
WO2001062800A1 (en) * 2000-02-24 2001-08-30 Eidgenössische Technische Hochschule Zürich Antibody specific for the ed-b domain of fibronectin, conjugates comprising said antibody, and their use for the detection and treatment of angiogenesis
MXPA04006517A (en) * 2002-01-03 2005-03-31 Schering Ag Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours.

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999058570A2 (en) * 1998-05-11 1999-11-18 Eidgenössische Technische Hochschule Zürich Antibodies to the ed-b domain of fibronectin, conjugates containing them and use therefor for diagnosis and therapy of tumors and diseases associated with angiogenesis

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SCHEUERMANN J.: "Isolation of binding molecules to the EDB domain offibronectin, a marker of angiogenesis", INTERNET CITATION, December 2002 (2002-12-01), Retrieved from the Internet <URL:http://e-collection.library.ethz.ch/eserv/eth:26198/eth-26198-02.pdf> [retrieved on 20110530] *
TARLI L. ET AL: "A high-affinity human antibody that targets tumoral blood vessels", BLOOD, vol. 94, no. 1, 1 July 1999 (1999-07-01), pages 192 - 198 *

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