Classifications

• • 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 [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], 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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