Title of Invention	"COMPOUND COMPRISING PEPTIDES DERIVED FROM RECOMBINANT ScFv ANTIBODY L19 AND AMINO ACID SEQUENCE FOR DIAGNOSIS AND TREATMENT OF TUMOR"
Abstract	The present invention relates to a compound that effectively binds radioisotopes and to extracellular ED-B domain of fibronectin for use in diagnosis and treatment of tumor comprising: a peptide derived from recombinant ScFv antibody L19 according to SEQ ID NO: 1 comprising: k) an antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity-determining regions HCDR3 (PFPYFDY) and/or LCDR3 (CQQTGRIPPT); or l) an antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity-determining regions HCDR1 (SFSMS), HCDR2 (SISGSSGTTYYADSVKG), HCDR3 (PFPYFDY), LCDR1 (RASQSVSSSFLA), LCDR2 (YASSRAT) and LCDR3 (CQQTGRIPPT); or m) a sequence according to Seq. Id. No. 1 (L19) and u) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2), wherein Xaa1, Xaa2 and Xaa3 each independently represent any naturally occuring amino acid or v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No. 3), wherein Xaa1, Xaa2, Xaa3, and Xaa4 each independently represent any naturally occuring amino acid or be) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6, wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond.

Title of Invention

"COMPOUND COMPRISING PEPTIDES DERIVED FROM RECOMBINANT ScFv ANTIBODY L19 AND AMINO ACID SEQUENCE FOR DIAGNOSIS AND TREATMENT OF TUMOR"

Abstract

The present invention relates to a compound that effectively binds radioisotopes and to extracellular ED-B domain of fibronectin for use in diagnosis and treatment of tumor comprising: a peptide derived from recombinant ScFv antibody L19 according to SEQ ID NO: 1 comprising: k) an antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity-determining regions HCDR3 (PFPYFDY) and/or LCDR3 (CQQTGRIPPT); or l) an antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity-determining regions HCDR1 (SFSMS), HCDR2 (SISGSSGTTYYADSVKG), HCDR3 (PFPYFDY), LCDR1 (RASQSVSSSFLA), LCDR2 (YASSRAT) and LCDR3 (CQQTGRIPPT); or m) a sequence according to Seq. Id. No. 1 (L19) and u) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2), wherein Xaa1, Xaa2 and Xaa3 each independently represent any naturally occuring amino acid or v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No. 3), wherein Xaa1, Xaa2, Xaa3, and Xaa4 each independently represent any naturally occuring amino acid or be) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6, wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond.

Full Text	The present invention relates to compound comprising peptides derived from recombinant ScFv antibody L19 and amino acid sequence for diagnosis and treatment of tumor. Brief description of the background art Tumours cannot gain more than a certain weight without the formation of new blood vessels (angiogenesis), and a correlation between microvessel density and tumour invasiveness has been reported for a number of tumours (Folkman (1995), Nature Med., 1, 27 - 31). Moreover, angiogenesis is involved in the majority of ocular disorders which result in loss of vision (Lee et al., Surv. Ophthalmol. 43, 245 - 269 (1998); Friedlander, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9764 - 9769 (1996)). Molecules capable of selectively targeting markers of angiogenesis would create clinical opportunities for the diagnosis and therapy of tumours and other diseases characterised by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration. Markers of angiogenesis are expressed in the majority of aggressive solid tumours in association with tumoural vessels and should therefore be readily accessible to specific binders injected intravenously (Pasqualini etal., (1997), Nature Biotechnoi., 15, 542-546; Neri et al. (1997), Nature Biotechnoi., 15, 1271 - 1275). Targeted occlusion of the neovasculature may result in tumour infarction and collapse (O'Reilly et al. (1996), Nature Med., 2, 689 - 692; Huang et al. (1997), Science, 275, 547 - 550). The ED-B domain of fibronectin, a sequence of 91 amino acids identical in mouse, rat and human, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around neo-vascular structures (Castellan! et al. (1994), Int. J. Cancer 59, 612 - 618) and could represent a target for molecular intervention. Indeed, it has recently been shown with fluorescent techniques that anti-ED-B single-chain Fv antibody fragments (scFv) accumulate selectively around tumoural blood vessels of tumour-bearing mice, and that antibody affinity appears to dictate targeting performance (Neri et al. (1997), Nature Biotechnol., 15, 1271 - 1275; WO 97/45544). Furthermore, antibodies and antibody fragments specific for binding the ED-B domain of fibronectin with a sub-nanomolar dissociation constant as well as radiolabeled derivatives thereof are described in WO 99/58570. The biodistribution of one of these high-affinity human antibody fragments, the 125I labelled antibody fragment called L19, was already investigated in tumour-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192 -198). Radiolabeled conjugates comprising L19-antibodies and their use for thej detection and treatment of angiogenesis are disclosed in WO 01/62800. The recombinant production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pas ton's has already been described (Marty etal., Protein Expression and Purification 21, 156 - 164 (2001)). Further, radiolabeling of scFv antibody fragments with "Tc through a C-terrninal cysteinyl peptide was described by George et al., Proc. Natl. Acad. ScL USA, Vol. 92 pp.. 8358 - 8362, 1995, and by Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868 - 872, 1996. However, there is still a clinical need for prividing antibody fragments that have improved pharmacokinetic properties, and that can easily be labeled with radioisotopes of e.g. Technetium or Rhenium, since these radionuclides are particular well suited for radiopharmaceuticals. Object of the invention It is therefore an object of the invention to provide antibody fragments that have improved pharmacokinetic properties, particularly target specifity and/or in vivo stability, and that can easily bind radioisotopes e.g. of Technetium or Rhenium. Summary of the invention The present invention describes compounds comprising a peptide comprising k) the sequence of the antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity- determining regions HCDR3 and/or LCDR3 as shown in Table 1 or a variation thereof that is a deletion, insertion and/or substitution of up to 5 arnino acids for the HCDR3 region and up to 6 amino acids for the LCDR3 region which has the same function as a peptide according to Seq. Id. No. 1; l) the sequence of the antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity- determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in Table 1 or a variation thereof that is a deletion, insertion and/or substitution of up to 3 amino acids for the HCDR1 region, up to 8 amino acids for the HCDR2 region, up to 5 amino acids for the HCDR3 region, up to 6 amino acids for the LCDR1 region, up to 4 amino acids for the LCDR2 region and up to 6 amino acids for the LCDR3 region; which has the same function as a peptide according to Seq. Id. No. 1; ac) the sequence according to Seq. Id. No. 1 (L19) or a variation of Seq. Id. No. 1 that is a deletion, insertion and/or substitution of up to 30 amino acids, and which has the same function as a peptide according to Seq. Id. No. 1, and u) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No, 2), wherein XaaT, Xaa2, and Xaa3 each independently represent any naturally occuring amino acid or v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4 (Seq. Id. No. 3), wherein Xaa,, Xaa2, Xaa3, and Xaa4 each independently represent any naturally occuring amino acid or be) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6, wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond. The compounds are preferably single chain antibody fragments, particularly scFv fragments. Further, the compounds are preferably conjugated to a radioisotope, e.g. a radioisotope of Technetium, such as 94mTc, 99mTo Rhenium, such as 186Re, 188Re, or other isotopes, such as 203Pb, 67Ga, 68Ga, 43Sc, 44Sc, 47Sc, 110mln, 111ln, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 90Y, 121Sn, 161Tb, 153Sm, 166Ho, 105Rh, 177Lu, 72As and 18F. The present invention also describes a pharmaceutical composition comprising the above compound as active agent together with physiologically acceptable adjuvants, diluents and/or carriers. The present invention also describes the use of a peptide comprising k) the sequence of the antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity- determining regions HCDR3 and/or LCDR3 as shown in Table 1 or a variation thereof that is a deletion, insertion and/or substitution of up to 5 amino acids for the HCDR3 region and up to 6 amino acids for the LCDR3 region which has the same function as a peptide according to SEQ Id. No. 1; l) the sequence of the antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity- -determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in Table 1 or a variation thereof that is a deletion, insertion and/or substitution of up to 3 amino acids for the HCDR1 region, up to 8 amino acids for the HCDR2 region, up to 5 amino acids for the HCDR3 region, up to 6 amino acids for the LCDR1 region, up to 4 amino acids for the LCDR2 region and up to 6 amino acids for the LCDR3 region; which has the same function as a peptide according to SEQ Id. No. 1; ac) a sequence according to Seq. Id. No. 1 (L19) or a variation of Seq. Id. No. 1 that is a deletion, insertion and/or substitution of up to 30 amino acids, and which has the same function as a peptide according to Seq. Id. No. 1, and u) an amino acid sequence XaarXaa2-Xaa3-Cys (Seq. Id. No. 2), wherein Xaau Xaa2, and Xaa3 each independently represent any naturally occuring amino acid or v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4 (Seq. Id. No. 3), wherein Xaa,, Xaa2, Xaa3, and Xaa4 each independently represent any naturally occuring amino acid or be) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6, wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond, for binding a radioisotope, e.g. a radioisotope of Technetium or Rhenium. The antibody fragment L19 is defined by the following sequence (Seq. Id. No. 1)(Sequence Removed) A deletion, insertion and/or substitution of up to 30 amino acids is a deletion, insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, I 2, 1 3, 1 4, 1 5, 1 6, 1 7, 18, 1 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids of Seq. Id. No.1. However, within the complementarity-determining regions (CDRs) the claimed peptide, e.g. the peptide of Seq. Id. No. 1, a variation that is a deletion, insertion and/or substitution of amino acids (aa) should not exceed the maximum variations defined in Table 1 below (HCDR: CDR of the heavy chain; LCDR: CDR of the light chain). (Table Removed)Table 1 The CDRs were defined according to E. A. Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, National Institutes for Health, Bethesda, MD, 5th Edition, 1991. Preferred are peptides comprising a sequence according to Seq. Id. No. 1 (L19) or a variation of Seq. Id. No. 1 that is a deletion, insertion and/or substitution of up to 20 amino acids. A peptide comprising a variation of the CDR sequences as shown in Table 1 and particularly a variation of Seq. Id. No. 1 that is a deletion, insertion and/or substitution, and which has the same function as the peptide according to Seq. Id. No. 1, is defined as a peptide that binds to the ED-B domain of fibronectin with a dissociation .constant Kd that is in the subnanomolar range {i.e. less than 10~9), measured with a BIAcore (see WO99/58570, Example 2 and Table 2). Preferred amino acid sequences Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2) are the sequences Gly-Gly-Gly-Cys (Seq. Id. No. 5) and Gly-Cys-Gly-Cys (Seq. id. No. 6). Most preferred is the sequence Gly-Gly-Gly-Cys (Seq. Id. No. 5). Preferred amino acid sequences Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No. 3) are the sequences Gly-Gly-Gly-Cys-Ala (Seq. Id. No. 7) and Gly-Cys-Gly-Cys-Ala (Seq. Id. No. 8). Most preferred is the sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No. 7). In compounds comprising an amino acid sequence (His)n (Seq. Id. No. 4), those compounds wherein n stands for the integer 6 are preferred. Preferred radioisotopes of Technetium or Rhenium are the isotopes 94mTc, 99mTc, 186Re and 188Re. Most preferred is the radioisotope 99mTc. Detailed Description of the Invention The single-chain antibody fragment L.19 (Seq. Id. No. 1) was previously labeled with 125I to investigate the biodistribution of this compound in tumour-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 1 92 -1 98). The results show that a selective targeting of tumoural blood vessels in vivo may be accomplished. Surprisingly however, it was found that the pharmacokinetic properties of the single-chain antibody fragment L1 9 may be substantially improved when it is conjugated to a peptide ba), bb) or be) and labelled with radioisotopes of Technetium or Rhenium. The isotope 99rnTc is the radiolabel of choice for routine clinical SPECT due to its radiochemical properties (easily available through a "Mo/"mTc generator, emits single gamma-photons of 140 KeV, has high photon flux, and decays with a half-life of 6 hours) and due to its cost-effectiveness. For therapeutic applications, labeling with the chemically analogous isotopes 1B6Re and 188Re is especially preferred (Hsieh, B.T., et al., Nucl. Med. Bioi., 1999, 26(8), 967-972; 973-976, Zarnora, P.O., et al., Anticancer Res., 1997, 17(3B), 1803-1838). The peptides of the present invention are derivatives of the recombinant scFv antibody L19 (Seq. Id. No. 1) against the extracellular ED-B domain of fibronectin and were produced via genetic engineering according to Fig. 1. The following peptides were produced: L19 (Seq. Id. No. i) (Sequence Removed) The production of the peptides is described in detail in the following examples (see ,,Experimentar). The antibody fragment L19 was originally produced by expression in E. coii (see WO 99/58570). However, for the large-scale production of scFv antibody fragments, this expression system was found to be unsatisfying. Another expression system, a yeast expression system, particularly a Pichia pastoris expression system, was tested. The present inventors found that yeast, e.g. Pichia pastoris is generally capable for expression of a highly bioactive antibody fragment, e.g. the fragment AP39, but a high yield expression with up to 250 mg antibody fragment per liter culture, which is necessary for an economical production of a biopharmaceutical, could only be reached by a constitutive expression vector (e.g. pGAP), and not with a methanol inducible vector (e.g. pPICSK). An additional advantage of this constitutive expression system is its simplified and robust fermentation procedures compared to an inducible yeast expression. Unexpectedly, the present inventors found that a proper signal sequence processing of the antibody fragment, e.g. the fragment AP39 was observed only when an expression cassette was used in which the N-terminus of the fragment was directly fused to the Kex2-cleavage site from the alpha-signal sequence. The peptides are suitable for diagnostic and therapeutic applications, particularly for the diagnosis and therapy of invasive tumours and tumour rnetastases. Preferred diagnostic applications are SPECT (Single Photon Emission Computed Tomography) and PET (Positron Emission Tomography). The peptides described above are particularly well suited for labeling radioisotbpes as described above, e.g. radioisotopes of Technetium and Rhenium, preferably the radionuclides 94mTc, 99mTc, 186Re, and 188Re. For labeling the peptides, the peptides are first reduced with an appropriate reducing agent like e.g. stannous chloride or Tris(2-carboxyethyl)phosphine (TCEP). The resulting reduced peptides exhibit SH-groups that can react with 99mTc generator eluate or 188Re generator eluate and stannous chloride to the compounds of the present: invention (for details, see the experimental examples below). Indirect labeling is performed by pre-conjugating a chelating ligand and subsequent complexation of radioisotopes, such as Indium, Yttrium, lanthanides etc. The chelating ligand is preferably derived from ethylene diamine tetraacetic acid (EDTA), diethylene triarnine pentaacetic acid (DTPA), cyclohexyl 1,2-diamine tetraaceticacid(CDTA),ethyleneglycol-0,0'-bis(2-aminoethyl)-N,N,N ',N ' iacetic acid (HBED), triethylene tetraamine hexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N"'-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), and 1,4,8,11-tetraazacyclotetradecane-N,f\T,N",N'"-tetr'aacetic acid (TETA), to either amine or thiol groups of the peptide compounds. The chelating ligands possess a suitable coupling group e.g. active esters, maleimides, thiocarbamates or. a-halogenated acetamide moieties. For conjugating chelating ligands to amine groups e.g. e-NH2-groups of lysine residues previous reduction of the peptide compounds is not required. The radiolabeled peptides are suitable for radio-diagnostic and radio-therapeutic applications. The resulting radiolabeled peptides show unexpected advantages in animal experiments. For example, excretion of a labeled peptide, e.g. 99mTc-labeled AP39 (Seq. Id. No. 11) in nude mice occurs to 70% or more, e.g. 80.63% within 24 hours via the kidneys, whereas for L19 (Seq. Id. No. 1) labeled with 125I, excretion in nude mice occured only to 67.79% via the kidneys within 24 hours. The tumour to blood ratio of a labeled peptide, e.g. 99mTc-labeled AP39 is 5:1 or more, preferably 8:1 or more, e.g. about 10:1 after 5 hours, whereas for LI 9 labeled with 125I, this ratio is only about 3:1. This is an unexpected behaviour also compared to other scFv antibodies labeled with 99mTc which often show less favourable biodistribution characteristics. For example, Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868 - 872, 1 996, report a ""To-labeled scFv antibody that shows a tumour to blood ratio of only 4 : 1 after 24 hours, and a kidney accumulation of 9% after 24 hours, which is very high compared to the values of the peptides described in the present invention, e.g. 1.3% for 99mTc-labeled AP39 (see example 13 below). Further, the in vivo stability of the labeled peptides of the invention, e.g. 99rTTc-labeled AP39 is much higher compared to the in vivo stability of L.1 9 labeled with 125I. The present inventors found that 2 hours after injection of a peptide, e.g. ""To-labeled AP39 only 10% or less, e.g. 3% of radioactivity within the serum was due to a metabolite, whereas 2 hours after injection of L19 labeled with ~25\, 49% of the radioactivity in the serum was due to metabolites, which may be free iodine. The improved in vivo stability of the peptides, e.g. 99mTc-labeled AP39 is also reflected by a prolonged preservation of its binding ability to the target ED-B. The present inventors found that 2 hours after injection of the peptide, e.g. 99mTc-labeled AP39, 50% or more, e.g. 74% of radioactivity within the serum was able to bind ED-B, whereas 2 hours after injection of 1-125 labeled L1 9, only 27% of radioactivity within the serum could bind to ED-B. The compounds of this invention are also showing high tumour accumulation. Forexample, Tc-99m-AP39 and ln-111 -MX-DTPA-e-HN(Lys)-AP39 displayed high tumour accumulation of 10.7 (Tc-99m ) or 12.9 (In-111) % injected dose per gram (ID/g) at 1 hour post injection (p.i.). Thus, tumor uptake is significantly higher compared to other known ln-111 or Tc-99m labeled antibody fragments (e.g. Kobayashi et al., J. Nuc. Med., Vol. 41 (4), pp. 755 - 762, 2000; Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868 - 872, 1996). The compounds are suitable for diagnostic and therapeutic applications. They are preferably applied to the patient by parenteral administration, more preferably by intravenous injection. The human dose is preferably in the range of 0.1 to 1 mg per patient for radiodiagnostic applications, and 0.1 to 10O mg per patient for radiotherapeutic applications. The methods for making and labeling the compounds of the present invention are more fully illustrated in the following examples. These examples are shown by way of illustration and not by way of limitation. Experimentai Example 1: Production of L19 derivatives A recombinant antibody (scFv L19, short name L19) against the extra domain B (ED-B) of a splice variant of fibronectin formed the starting material. scFv L19 had been isolated by means of phage display selection from a synthetic human antibody repertoire (Neri et al., 1997, Nature Biotechnol. 15: 1271; Pini etal.,-1998, J. Biol. Chem. 273: 21769). This recombinant antibody fragment is in the form of a so-called single chain antibody fragment (scFv) and consists of a VH and VL region connected by a linker sequence (see Seq. Id. No. 1). This scFv L19 has exceptionally high affinity for ED B (Kd: 5.4 x 1Q-11 M). Various derivatives of L19 were produced by genetic manipulation (see Fig. 1). To modify L19, the scFv encoding DNA was amplified by PCR (polymerase chain reaction) using primers which coded for the additional sequences, and cloned into expression vectors. L19 derivatives: L19: without additional terminal modifications LI 9 His: C-terminal His6 domain (His tag), for Ni chelate chrornatography and for binding radioisotopes AP38: C-terminal GlyGlyGlyCys domain for binding (via Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes) AP39: C-terminal GlyGlyGlyCysAla domain for binding (via Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes) L19-GlyCysGlyCys: C-terminal GlyCysGlyCys domain for binding (via Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes) L19-GlyCysGlyCysAla: C-terminal GlyCysGlyCysAla domain for binding (via Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes) Recombinant production of LI9 derivatives The L19 derivatives described were produced in prokaryotic and eukaryotic expression systems. a) LI 9 production in E. coli The DMA sequences encoding various L19 derivatives (AP38, AP39, L19-GlyCysGlyCys, L19-GlyCysGlyCysAla, L19, L19His) were cloned into a prokaryotic expression vector (pDN5, Pini et al., 1997, J. Immunol, Methods 206: 171, Pini et al., 1998, J. Biol. Chem. 273: 21769; pET, Novageh) with IPTG-inducible promoter and ampicillin resistance marker. In order to make secretion of the recombinant protein into the periplasm possible, this vector was used to produce an expression cassette in which the N terminus of scFv is fused to a Pel B signal sequence. It was possible to establish stable producer strains by transforming E. coli (TG1, BL21DE3 and HB2151) with this expression vector, followed by ampicillin selection. To produce scFv, these strains were cultivated in the presence of 1 % glucose in the growth phase (37°C) in order to repress the promoter. Expression of scFv in the cultures was induced by adding IPTG and incubating at 30°C for up to 16h. Soluble and antigen-binding scFv material could be isolated from the complete extract of the E. coli strains, from the periplasm fraction or, which proved to be particularly efficient in relation to purification and yield, from the culture supernatant. Production took place in shaken flasks and in fermenters with a culture volume of up to 10 litres. b) Production of LI 9 derivatives in Pichia pastoris L19His, AP38, AP39, L19-GlyCysGryCys and L1 9-GlyCysGlyCysAla-encoding DNA sequences were amplified by PCR and cloned into E. coli and into the expression vectors pPIC9K and pGAP (Invitrogen) for production in the yeast Pichia pastoris. For expression of heterologous genes, pPIC9K contains a methanol-inducible promoter (AOX1), and pGAP contains the constitutive promoter of the GAPDH enzyme. In addition, these vectors contain respectively a geneticin resistance gene and a zeocin resistance gene for selection/amplification of the foreign gene and a signal sequence (from yeast a factor) for expression and secretion of the recombinant product. The AP39 expression cassette used codes for a fusion protein (a factor signal + L19 derivatives) which contains for signal sequence elimination only a Kex2 cleavage site and not the other cleavage sites of natural a factor processing. Stable transfected PP clones were established by electroporation of the linearized vectors into Pichia pastoris strains (e.g. pPIC9K-AP39 into strain GS115, pGAP^-AP39 into strain X33) and subsequent geneticin or zeocin selection. It was possible to use these clones to produce the said L19 derivatives as soluble secretory protein. The clones were cultivated at 30°C in BMGY medium or basal mineral medium. With clones based on pPIC, methanol was added for promoter induction during the expression phase. The recombinant product had a correctly processed terminus and high antigen-binding activity. The yields which could be achieved (unpurified, bioactive product/litre of culture supernatant) were, depending on the culturing conditions and process control: e.g. pPIC9K-AP39/GS115 (shaken flask 5 mg/l, fermenter 10-15mg/l); pGAP-AP39/X33 (shaken flask 30-40 mg/l, fermenter 100 250 mg/l). The L19 derivatives were purified from the Pichia pastoris or E, coli culture supernatant by use of affinity chromatography (rProtein A, Streamline Pharmacia or ED B antigen column) with subsequent size exclusion chromatography. The purified AP39 fraction, which was employed for further processing, had a homodimer structure (with subunits covalently linked for the most part) and high antigen-binding activity. Example 2a Synthesis of reduced AP38 [Reduced L19-(Gly)3-Cys-OH] To a solution of 240 /yg (4.29 nmol) S-S-dimeric AP38 in 156 fj\ PBS {phosphate buffered saline)/! 0% glycerine were added 50//I TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na2HPO4, 0.1 M, pH = 7.4),. The reaction mixture was gently shaken for 1 h at room temperature. SH-tnonomeric AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S-S-dimeric AP38 to SH-rnonomeric AP38. Yield: 79.4 /yg/220 jj\ PBS (33.1%). Example 2b Synthesis of Tc-99m-AP38 [Tc-99m-L19-(Gly)3-Cys-OH] 2,37 mg disodium-L-tartrate were placed in a vial followed by addition of 79.4 //g reduced AP38 in 220 //I PBS and the solution was diluted with 100 fj\ aqueous Na2HP04-buffer (1 M, pH = 10.5). 50 A/I Tc-99m generator eluate (24 h) and 10//I SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 0.5 h at 37°C. Tc-99m-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 39.7%. Radiochemical purity: 92.5% (SDS-PAGE). Specific activity: 17.7 MBq/nmol. Immunoreactivity: 88.7% Example 3a Synthesis of reduced AP39 [Reduced L19-(Gly)3-Cys-Ala-OH] To a solution of 240//g (4.29 nmol) S-S-dimeric AP39 in 135/vl PBS/10% glycerine were added 50 //I TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na2HPO4, 0.1 M, pH = 7.4). The reaction mixture was gently shaken for 1 h-at room temperature. SH-monomeric AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S.-S-dimeric AP39 to SH-monomeric AP39. Yield: 135.9 //g/1 80 u\ PBS (56.2%). Example 3b Synthesis of Tc-99m-AP39 [Tc-99m-L19-(GIy)3-Cys-Ala-OH] 4.2 mg disodium-L-tartrate were placed in a vial followed by addition of 135.9 fjg reduced AP39 in 180 //I PBS and the solution was diluted with 100 //I aqueous Na2HPO4-buffer (1 M, pH = 10.5). 100 jj\ Tc-99m generator eluate (24 h) and 10//I SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 0.5 h at 37°C. Tc-99m-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 50.1%. Radiochemical purity: 91.5% (SDS-PAGE). Specific activity: 21.4 MBq/nmol. Irnmunoreactivity: 96.4% Example 4 Synthesis of Re-188-AP38 [Re-188-L19-(Gly)3-Cys-OH] 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 1 12>L/g reduced AP38 in 310/y\| PBS and the solution was diluted with 100 /j\ aqueous Na2HPO4-buffer (1 M, pH = 10.5). 100//I Re-188 generator eluate and 50 jj\ SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 1.5 h at 37°C. Re-188-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 28.3%. Radiochemical purity: Specific activity: Immune-reactivity:91.1% -(SOS-PAGE). 15.3 MBq/nmol. 89.9% Example 5 Synthesis of Re-188-AP39 [Re-188-L19-(Gly)3-Cys-Ala-OH] 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 1 12/yg reduced AP39 in 303//I PBS and the solution was diluted with 100 //I aqueous Na2HPO4-buffer (1 M, pH = 10.5). 100 fj\ Re-188 generator eluate and 50/vl SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 1.5 h at 37°C. Re-188-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: Radiochemical purity: Specific activity: Immunoreactivity: 33.5%. 92.3% .(SOS-PAGE). 18.5 MBq/nmol. 92.5% Example 6a Synthesis of reduced L19-Gly-Cys-Gly-Cys-OH To a solution of 240 //g (4.29 nmol) S-S-dimeric L19-Gly-Cys-Gly-Cys-OH in 160//I PBS/10% glycerine were added 75 //I TCEP-solution (14.34 rng TCEP x HCI/5 ml aqueous Na2HP04, 0.1 M, pH - 7.4). The reaction mixture was gently shaken for 1 h at room temperature. SH-monomeric L1 9-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S-S-dimeric L19-Giy-Cys-Gly-Cys-OH to SH-monomeric L1 9-Gly-Cys-Gly-Cys-OH. Yield: 80.4//g/210//I PBS (33.5%). Example 6b Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-OH 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 80.4/yg reduced L19-Gly-Cys-Gly-Cys-OH in 210/yl PBS and the solution was diluted with 100//I aqueous Na2HPO4-buffer (1 M, pH = 10.5). 50/vl Tc-99m generator eluate (24 h) and 10 fj\ SnCI2-solution (5 mg SnCI2/l rnl 0.1 M HCI) were added. The reaction mixture was shaked for 0.5 h at 37°C. Tc-99m-labeled L.19-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 37.7%. Radiochemical purity: 91.5% (SDS-PAGE). Specific activity: 19.7 MBq/nmol. Irnmunoreactivity: 89.7% Example 7a Synthesis of reduced L19-Gly-Cys-Gly-Cys-AIa-OH To a solution of 240//g (4.29 nmol) S-S-dimeric L19-Gly-Cys-Gly-Cys-Ala-OH in 155 //I PBS/10% glycerine were added 75//I TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na2HPO4, 0.1 M, pH = 7.4). The reaction mixture was gently snaked for 1 h at room temperature. SH-monomeric L1 9-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S-S-dimeric L19-Gly-Cys-Gly-Cys-Ala-OH to SH-monomeric L19-Gly-Cys-Gly-Cys-Ala-OH. Yield: 81.2//g/215//I PBS (33.8%). Example 7b Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-Ala-OH 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 81.2 jjg reduced L19-Gly-Cys-Gly-Cys-Ala-OH in 215 //I PBS and the solution was diluted with 100 jj\ aqueous Na2HPO4-buffer (1 M, pH = 10.5). 50/ylTc-99m generator eluate (24 h) and 10//I SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaked for 0.5 h at 37°C. Tc-99m-labeled L19-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 35.6%. Radiochernical purity: 93.5% (SDS-PAGE). Specific activity: 19.1 MBq/nmol. Immunoreactivity: 88.7% Example 8a Synthesis of reduced AP39 for specific conjugation of EDTA, CDTA, TETA, DTPA, TTHA, HBED, DOTA, NOTA, and DO3A type chelators to the Cysteine-SH group 50//I TCEP-solution (14.34mg TCEPxHCI/5ml aqueous Na2HPO4/ 0.1M, pH = 7.4) were added to a solution of 400/yg (7.1 nmol) AP39 in 450//I PBS. The reaction mixture was gently shaken for 1h at 37 °C. Reduced AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: sodium acetate buffer, 0.1M, pH 5.0). SDS-PAGE analysis of the isolated product proofed the complete? transformation of AP39 into reduced AP39. Yield: 140//g/200//l (35%). Example 8b Synthesis of MX-DTPA-Maleimide (1,4,7-triaza-2-{N-maleimido ethylene/?-amino)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl 6-methyl heptane) 512 mg (1 mmol) of {[3-(4-Arnino-phenyl)-2-(bis-carboxymethyl-amino)-propyl]-[2-(bis-carboxymethyl-amino)-propyl]-amino}-acetic acid (Macrocyclics Inc. Dallas, TX, U.S.A.) and 707 mg (7 mmol) triethylarnine were dissolved in 3 ml dry DMF. 400 mg (1,5 mmol) of 3-(2,5-Dioxo-2,5-d)hydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Aldrich) in 1 ml dry DMF were added dropwisely. The solution was stirred for 5 h at 50° C. 30 ml of diethylether were added slowly. The reaktion mixture was stirred for further 30 min. The principate was collected by filtering. The crude product was purified by RP-HPLC (acetonitrile- : water- : trifluoracetic acid / 3 : 96,9 : 0,1 -*• 99,9 : 0 : 0,1). Yield: 61% (405 mg, 0,61 mmol). MS-ESI: 664 = M+ +1. Example 8c Synthesis of ln-111-MX-DTPA-MaIeirnide-S{Cys)-AP39-R (R = reduced) 140/yg (5 nmol) AP39-R in 200 /j\ of sodium acetate buffer (0.1M, pH 5) were reacted with 50/c/l of dissolved 1,4,7-triaza-2-(N-maleimido ethylene p-amino) benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl6-methyl heptane (0,25mg DTPA-Maleimide in 500//I sodium acetate buffer 0.1M pH 5) for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h with 200rnl of sodium acetate buffer (0.1 M, pH 6) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 80 p\ [ln-1 11 ]lnCI3 solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37 °C for 30 min. ln-111 labeled DTPA-Maleimide-S(Cys)-AP39-R was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 54 %. Radiochemical purity: 94 % (SDS-PAGE). Specific activity: 6.2 MBq/nmol. Immunoreactivity: 86 % Example 9 Synthesis of ln-111-MX-DTPA-e-HN(Lys)-AP3£ 200//g (3.6 nmol) non-reduced AP39 in 111 /j\ PBS were diluted with 300 fj\ of sodium borate buffer (0.1M, pH 8.5} and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 50 //I of 1,4,7~triaza-2-(p-isothiocyanato) benzyl- 1,7-bis(carboxyrriethyl)-4-carboxymethyl-6-rnethyl heptane (MX-DTPA) solution (0.33 mg MX-DTPA dissolved in 500 //I of sodium borate buffer, 0.1M, pH 8.5) were added and the reaction mixture was heated for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h and 1 x 1 7 h (over night) with 200 ml of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 80 A/I [ln-1 11]lnCI3 solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37°G for 30 min. ln-111 labeled MX-DTPA-e-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 70 %. Radiochemical purity: 85 % (SDS-PAGE). Specific activity: 7.6 MBq/nmol. Irnmunoreactivity: 74 % Example 10 Synthesis of ln-111 -DOTA-C-Benzyl-p-NCS -e-HN(Lys)-AP39 200//g (3.6 nmol) non-reduced AP39 in 114//I PBS were diluted with 300 fj\ of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc.,. Rockford, IL, U.S.A.). 50//I of 1,4,7,1O-tetraaza-2-i isothiocyanato)benzyl cyclododecane-1,4,7,10-tetraacetic acid (benzyl-p-SCN-DOTA, Macrocyclics Inc., Dallas TX, U.S.A.) solution (1.5 mg benzyl-p-SCN-DOTA dissolved in 5 ml of sodium borate buffer, 0.1M, pH 8.5) were added to the solution and the reaction mixture was heated for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h and 1 x 17 h (over night) with 200 ml of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 80 fj\ [ln-111 ]lnCI3 solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37°C for 30 min. ln-111 labeled DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 74 %. Radiochemical purity: 94 % (SDS-PAGE). Specific activity: 12.3 MIBq/nmol. Immunoreactivity: 73 % Example 11 Synthesis of Y-88-MX-DTPA-e-HN(Lys)-AP39 200 jug (3.6 nmol) non-reduced AP39 in 115//I PBS were diluted with 300 //I of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,OOO MWCO (Pierce Inc., Rockford, IL, U.S.A.). 50 //I of MX-DTPA solution (0.33 mg MX-DTPA dissolved in 500/vl of sodium borate buffer, 0.1M, pH 8.5) were added and the reaction mixture was heated for 3 h at 37 ° C. The reaction mixture was dialyzed 2 x 1 h and 1 x 17 h (over night) with 200 rnl of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 100 //I [Y-88]YCI3 solution (HCI, 1N, 75 MBq, Oak Ridge National Lab.) were added and the reaction mixture was heated at 37°C for 30 min. Y-88 labeled MX-DTPA-«r-HN(LyshAP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). Radiochemical yield: 65 %. Radiochemical purity: 93 % (SDS-PAGE). Specific activity: 10.2 MBq/nmol. Irnmunoreactivity: 72 % Example 12 Synthesis of Lu-177 -DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP3 200/vg (3.6 nmol) non-reduced AP39 in 110//I PBS were diluted with 300 //I of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 50//I of benzyl-p-SCN-DOTA solution {1.5 mg dissolved in 5 ml of sodium borate buffer, 0.1M, pH 8.5) were added and the reaction mixture was heated for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h and 1 x 1 7 h (over night) with 200 ml of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 200//I [Lu-177]LuCI3 solution (HCI, 1N, 80MBq, NRH-Petten, Netherlands) were added and the reaction mixture was heated at 37 °C for 30 min. Lu-177 labeled DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP39 was purified by gel-chromatography using.a NAP-5-column (Amersham, Eluent: PBS). Radiochemical yield: 74 %. Radiochemical purity: 95 % (SDS-PAGE). Specific activity: 19 MBq/nmol. Immunoreactivity: 71 % Example 13 Organ distribution and excretion of Tc-99m-AP39, expressed in Pichia pastoris, after a single i.v. injection into tumour-bearing nude mice The substance of the invention is injected intravenously in a dose of about 74 kBq into F9 (teratocarcinoma)-bearing animals (bodyweightabout25 g). The radioactivity concentration in various organs, and the radioactivity in the excreta are measured using a y counter at various times after administration of the substance. In addition, the tumour to blood ratio is found at various times on the basis of the concentration of the substance of the invention in tumour and blood, The biodistribution of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice {mean ± SD, n = 3) is shown in Table 2: (Table Removed) Table 2 The excretion of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Table 3: (Table Removed)Table 3 The tumour to blood ratio of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Fig. 2. The results of this investigation show the excellent potential of the substance of the invention for accumulation in solid tumours with, at the same time, excellent excretion. Example 14 Organ distribution of ln-111-MX-DTPA- The substance of the invention is injected intravenously in a dose of about 48 kBq into F9 (teratocarcinoma)-bearing animals (body weight about 25 g). The radioactivity concentration in various organs, and the radioactivity in the excreta are measured using a y counter at various times after administration of the substance, The biodistribution of ln-111-MX-DTPA-e-HN(Lys)-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Table 4: (Table Removed)Table 4 The tumour to blood ratio of ln-111-MX-DT.PA-e-HN(l_ys)-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Table 5. (Table Removed)Table 5 The results of this investigation show the excellent potential of the substance of the invention for accumulation in solid tumours paired with excellent biodistribution and tumor to blood ratio. Example 15 Imaging of Tc-99m-AP39, expressed in Pichia pastoris, after a single i.v. injection into tumour-bearing nude mice The substance of the invention is injected intravenously in a dose of about 9.25 MBq into F9 (teratocarcinoma)-bearing animals {bodyweight about 25 g). Gamma-camera imaging is carried out at various times after administration of the substance. Planar scintigraphy of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice is shown in Figures 3 and 4. Fig. 3 shows the scintigram 5 hours after injection of the substance, and Fig. 4 shows the scintigram 24 hours after injection of the substance. The result of this investigation shows the excellent potential of the substance of the invention for imaging solid tumours. WE CLAIM: 1. A compound that effectively binds to radioisotopes and to extracellular ED-B domain of fibronectin for use in diagnosis and treatment of tumor comprising: a peptide derived from recombinant ScFv antibody L19 according to SEQ ID NO: 1 comprising: k) an antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity-determining regions HCDR3 (PFPYFDY) and/or LCDR3 (CQQTGRIPPT); or l) an antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity-determining regions HCDR1 (SFSMS), HCDR2 (SISGSSGTTYYADSVKG), HCDR3 (PFPYFDY), LCDR1 (RASQSVSSSFLA), LCDR2 (YASSRAT) and LCDR3 (CQQTGRIPPT); or m) a sequence according to Seq. Id. No. 1 (L19) and u) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2), wherein Xaa1, Xaa2 and Xaa3 each independently represent any naturally occuring amino acid or v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No. 3), wherein Xaa1, Xaa2, Xaa3, and Xaa4 each independently represent any naturally occuring amino acid or bc) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6, wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond. 2. The compound as claimed in claim 1, wherein the amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2) is the sequence Gly-Gly-Gly-Cys (Seq. Id. No. 5) or Gly-Cys-Gly-Cys (Seq. Id. No. 6). 3. The compound as claimed in claim 1, wherein the amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4 (Seq. Id. No. 3) is the sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No. 7) or Gly-Cys-Gly Cys-Ala (Seq. Id. No. 8). 4. The compound as claimed in claim 1, wherein n in the amino acid sequence (His)n (Seq. Id. No. 4) is 6. 5. The compound as claimed in any one of claims 1 to 4 which is optionally conjugated to a radioisotope. 6. The compound as claimed in claim 6 which is conjugated to a radioisotope selected from a radioisotope of Technetium, such as 94mTc, 99mTc, Rhenium, such as I86Re, 188Re, or other isotopes, such as 203Pb, 67Ga, 68Ga, 43Sc, ^Sc, 47Sc, 110mIn, 111In, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 90Y, 121Sn, 161Tb, 153Sm, 166Ho, 105Rh, 177Lu, 72As and 18F. 7. A compound as claimed in claim 6, wherein the radioisotope is 99mTc or 188Re. 8. The compound as claimed in any one of claims 1 to 7, wherein the peptide is in reduced form.

Full Text

The present invention relates to compound comprising peptides derived from recombinant ScFv antibody L19 and amino acid sequence for diagnosis and treatment of tumor.
Brief description of the background art
Tumours cannot gain more than a certain weight without the formation of new blood vessels (angiogenesis), and a correlation between microvessel density and tumour invasiveness has been reported for a number of tumours (Folkman (1995), Nature Med., 1, 27 - 31). Moreover, angiogenesis is involved in the majority of ocular disorders which result in loss of vision (Lee et al., Surv. Ophthalmol. 43, 245 - 269 (1998); Friedlander, M. et al., Proc. Natl. Acad. Sci. U.S.A. 93, 9764 - 9769 (1996)). Molecules capable of selectively targeting markers of angiogenesis would create clinical opportunities for the diagnosis and therapy of tumours and other diseases characterised by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration. Markers of angiogenesis are expressed in the majority of aggressive solid tumours in association with tumoural vessels and should therefore be readily accessible to specific binders injected intravenously (Pasqualini etal., (1997), Nature Biotechnoi., 15, 542-546; Neri et al. (1997), Nature Biotechnoi., 15, 1271 - 1275). Targeted occlusion of the neovasculature may result in tumour infarction and collapse (O'Reilly et al. (1996), Nature Med., 2, 689 - 692; Huang et al. (1997), Science, 275, 547 - 550).
The ED-B domain of fibronectin, a sequence of 91 amino acids identical in mouse, rat and human, which is inserted by alternative splicing into the fibronectin molecule, specifically accumulates around neo-vascular structures (Castellan! et al. (1994), Int. J. Cancer 59, 612 - 618) and could represent a target for molecular intervention. Indeed, it has recently been shown with fluorescent techniques that anti-ED-B single-chain Fv antibody fragments (scFv) accumulate selectively around tumoural blood vessels of tumour-bearing mice, and that antibody affinity appears to dictate targeting performance (Neri et al. (1997), Nature Biotechnol., 15, 1271 - 1275; WO 97/45544).
Furthermore, antibodies and antibody fragments specific for binding the ED-B domain of fibronectin with a sub-nanomolar dissociation constant as well as radiolabeled derivatives thereof are described in WO 99/58570. The biodistribution of one of these high-affinity human antibody fragments, the 125I labelled antibody fragment called L19, was already investigated in tumour-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192 -198). Radiolabeled conjugates comprising L19-antibodies and their use for thej detection and treatment of angiogenesis are disclosed in WO 01/62800.
The recombinant production of functionalized single-chain Fv antibody fragments binding to the ED-B domain of the B-isoform of fibronectin in Pichia pas ton's has already been described (Marty etal., Protein Expression and Purification 21, 156 - 164 (2001)).
Further, radiolabeling of scFv antibody fragments with "Tc through a C-terrninal cysteinyl peptide was described by George et al., Proc. Natl. Acad. ScL USA, Vol. 92 pp.. 8358 - 8362, 1995, and by Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868 - 872, 1996.
However, there is still a clinical need for prividing antibody fragments that have improved pharmacokinetic properties, and that can easily be labeled with radioisotopes of e.g. Technetium or Rhenium, since these radionuclides are particular well suited for radiopharmaceuticals.
Object of the invention
It is therefore an object of the invention to provide antibody fragments that have improved pharmacokinetic properties, particularly target specifity and/or in vivo stability, and that can easily bind radioisotopes e.g. of Technetium or Rhenium.
Summary of the invention
The present invention describes compounds comprising a peptide comprising
k) the sequence of the antigen-binding site for the extra domain
B (ED-B) of fibronectin comprising complementarity-
determining regions HCDR3 and/or LCDR3 as shown in Table
1 or a variation thereof that is a deletion, insertion and/or
substitution of up to 5 arnino acids for the HCDR3 region and
up to 6 amino acids for the LCDR3 region which has the same
function as a peptide according to Seq. Id. No. 1;
l) the sequence of the antigen-binding site for the extra domain
B(ED-B) of fibronectin comprising complementarity-
determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2
and LCDR3 as shown in Table 1 or a variation thereof that is
a deletion, insertion and/or substitution of up to 3 amino acids
for the HCDR1 region, up to 8 amino acids for the HCDR2
region, up to 5 amino acids for the HCDR3 region, up to 6
amino acids for the LCDR1 region, up to 4 amino acids for the LCDR2 region and up to 6 amino acids for the LCDR3 region; which has the same function as a peptide according to Seq. Id. No. 1;
ac) the sequence according to Seq. Id. No. 1 (L19) or a variation of Seq. Id. No. 1 that is a deletion, insertion and/or substitution of up to 30 amino acids, and which has the same function as a peptide according to Seq. Id. No. 1,
and
u) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No, 2),
wherein XaaT, Xaa2, and Xaa3 each independently represent
any naturally occuring amino acid or
v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4 (Seq. Id.
No. 3), wherein Xaa,, Xaa2, Xaa3, and Xaa4 each
independently represent any naturally occuring amino acid or
be) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6,
wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond.
The compounds are preferably single chain antibody fragments, particularly scFv fragments. Further, the compounds are preferably conjugated to a radioisotope, e.g. a radioisotope of Technetium, such as 94mTc, 99mTo Rhenium, such as 186Re, 188Re, or other isotopes, such as 203Pb, 67Ga, 68Ga,
43Sc, 44Sc, 47Sc, 110mln, 111ln, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 90Y, 121Sn, 161Tb, 153Sm, 166Ho, 105Rh, 177Lu, 72As and 18F.
The present invention also describes a pharmaceutical composition comprising the above compound as active agent together with physiologically acceptable adjuvants, diluents and/or carriers.
The present invention also describes the use of a peptide comprising
k) the sequence of the antigen-binding site for the extra domain
B (ED-B) of fibronectin comprising complementarity-
determining regions HCDR3 and/or LCDR3 as shown in Table
1 or a variation thereof that is a deletion, insertion and/or
substitution of up to 5 amino acids for the HCDR3 region and
up to 6 amino acids for the LCDR3 region which has the same
function as a peptide according to SEQ Id. No. 1;
l) the sequence of the antigen-binding site for the extra domain
B(ED-B) of fibronectin comprising complementarity-
-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 as shown in Table 1 or a variation thereof that is a deletion, insertion and/or substitution of up to 3 amino acids for the HCDR1 region, up to 8 amino acids for the HCDR2 region, up to 5 amino acids for the HCDR3 region, up to 6 amino acids for the LCDR1 region, up to 4 amino acids for the LCDR2 region and up to 6 amino acids for the LCDR3 region; which has the same function as a peptide according to SEQ Id. No. 1;
ac) a sequence according to Seq. Id. No. 1 (L19) or a variation of
Seq. Id. No. 1 that is a deletion, insertion and/or substitution
of up to 30 amino acids, and which has the same function as a peptide according to Seq. Id. No. 1,
and
u) an amino acid sequence XaarXaa2-Xaa3-Cys (Seq. Id. No. 2),
wherein Xaau Xaa2, and Xaa3 each independently represent
any naturally occuring amino acid or
v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4 (Seq. Id.
No. 3), wherein Xaa,, Xaa2, Xaa3, and Xaa4 each
independently represent any naturally occuring amino acid or
be) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6,
wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond,
for binding a radioisotope, e.g. a radioisotope of Technetium or Rhenium.
The antibody fragment L19 is defined by the following sequence (Seq. Id.
No. 1)(Sequence Removed)
A deletion, insertion and/or substitution of up to 30 amino acids is a deletion, insertion and/or substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, I 2, 1 3, 1 4, 1 5, 1 6, 1 7, 18, 1 9, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids of Seq. Id. No.1. However, within the complementarity-determining regions (CDRs) the claimed peptide, e.g. the peptide of Seq. Id. No. 1, a variation that is a deletion, insertion and/or substitution of amino acids (aa) should not exceed the maximum variations defined in Table 1 below (HCDR: CDR of the heavy chain; LCDR: CDR of the light chain).

(Table Removed)Table 1
The CDRs were defined according to E. A. Kabat et al., "Sequences of Proteins of Immunological Interest", U.S. Department of Health and Human Services, National Institutes for Health, Bethesda, MD, 5th Edition, 1991.
Preferred are peptides comprising a sequence according to Seq. Id. No. 1 (L19) or
a variation of Seq. Id. No. 1 that is a deletion, insertion and/or substitution of up to 20 amino acids.
A peptide comprising a variation of the CDR sequences as shown in Table 1 and particularly a variation of Seq. Id. No. 1 that is a deletion, insertion
and/or substitution, and which has the same function as the peptide according to Seq. Id. No. 1, is defined as a peptide that binds to the ED-B domain of fibronectin with a dissociation .constant Kd that is in the subnanomolar range {i.e. less than 10~9), measured with a BIAcore (see WO99/58570, Example 2 and Table 2).
Preferred amino acid sequences Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2) are the sequences Gly-Gly-Gly-Cys (Seq. Id. No. 5) and Gly-Cys-Gly-Cys (Seq. id. No. 6). Most preferred is the sequence Gly-Gly-Gly-Cys (Seq. Id. No. 5).
Preferred amino acid sequences Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No. 3) are the sequences Gly-Gly-Gly-Cys-Ala (Seq. Id. No. 7) and Gly-Cys-Gly-Cys-Ala (Seq. Id. No. 8). Most preferred is the sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No. 7).
In compounds comprising an amino acid sequence (His)n (Seq. Id. No. 4), those compounds wherein n stands for the integer 6 are preferred.
Preferred radioisotopes of Technetium or Rhenium are the isotopes 94mTc, 99mTc, 186Re and 188Re. Most preferred is the radioisotope 99mTc.
Detailed Description of the Invention
The single-chain antibody fragment L.19 (Seq. Id. No. 1) was previously labeled with 125I to investigate the biodistribution of this compound in tumour-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 1 92 -1 98). The results show that a selective targeting of tumoural blood vessels in vivo may be accomplished. Surprisingly however, it was found that the pharmacokinetic properties of the single-chain antibody fragment L1 9 may be substantially improved when it is conjugated to a peptide ba), bb) or be) and labelled with radioisotopes of Technetium or Rhenium. The isotope 99rnTc is the radiolabel of choice for routine clinical SPECT due to its
radiochemical properties (easily available through a "Mo/"mTc generator, emits single gamma-photons of 140 KeV, has high photon flux, and decays with a half-life of 6 hours) and due to its cost-effectiveness. For therapeutic applications, labeling with the chemically analogous isotopes 1B6Re and 188Re is especially preferred (Hsieh, B.T., et al., Nucl. Med. Bioi., 1999, 26(8), 967-972; 973-976, Zarnora, P.O., et al., Anticancer Res., 1997, 17(3B), 1803-1838).
The peptides of the present invention are derivatives of the recombinant scFv antibody L19 (Seq. Id. No. 1) against the extracellular ED-B domain of fibronectin and were produced via genetic engineering according to Fig. 1. The following peptides were produced:
L19 (Seq. Id. No. i) (Sequence Removed)
The production of the peptides is described in detail in the following examples (see ,,Experimentar).
The antibody fragment L19 was originally produced by expression in E. coii (see WO 99/58570). However, for the large-scale production of scFv antibody fragments, this expression system was found to be unsatisfying. Another expression system, a yeast expression system, particularly a Pichia pastoris expression system, was tested. The present inventors found that yeast, e.g. Pichia pastoris is generally capable for expression of a highly bioactive antibody fragment, e.g. the fragment AP39, but a high
yield expression with up to 250 mg antibody fragment per liter culture, which is necessary for an economical production of a biopharmaceutical, could only be reached by a constitutive expression vector (e.g. pGAP), and not with a methanol inducible vector (e.g. pPICSK). An additional advantage of this constitutive expression system is its simplified and robust fermentation procedures compared to an inducible yeast expression. Unexpectedly, the present inventors found that a proper signal sequence processing of the antibody fragment, e.g. the fragment AP39 was observed only when an expression cassette was used in which the N-terminus of the fragment was directly fused to the Kex2-cleavage site from the alpha-signal sequence.
The peptides are suitable for diagnostic and therapeutic applications, particularly for the diagnosis and therapy of invasive tumours and tumour rnetastases. Preferred diagnostic applications are SPECT (Single Photon Emission Computed Tomography) and PET (Positron Emission Tomography).
The peptides described above are particularly well suited for labeling radioisotbpes as described above, e.g. radioisotopes of Technetium and Rhenium, preferably the radionuclides 94mTc, 99mTc, 186Re, and 188Re. For labeling the peptides, the peptides are first reduced with an appropriate reducing agent like e.g. stannous chloride or Tris(2-carboxyethyl)phosphine (TCEP). The resulting reduced peptides exhibit SH-groups that can react with 99mTc generator eluate or 188Re generator eluate and stannous chloride to the compounds of the present: invention (for details, see the experimental examples below). Indirect labeling is performed by pre-conjugating a chelating ligand and subsequent complexation of radioisotopes, such as Indium, Yttrium, lanthanides etc. The chelating ligand is preferably derived from ethylene diamine tetraacetic acid (EDTA), diethylene triarnine pentaacetic acid (DTPA), cyclohexyl 1,2-diamine tetraaceticacid(CDTA),ethyleneglycol-0,0'-bis(2-aminoethyl)-N,N,N ',N '
iacetic acid (HBED), triethylene tetraamine hexaacetic acid (TTHA), 1,4,7,10-tetraazacyclododecane-N,N',N"'-tetraacetic acid (DOTA), 1,4,7-triazacyclononane-N,N',N"-triacetic acid (NOTA), and 1,4,8,11-tetraazacyclotetradecane-N,f\T,N",N'"-tetr'aacetic acid (TETA), to either amine or thiol groups of the peptide compounds. The chelating ligands possess a suitable coupling group e.g. active esters, maleimides, thiocarbamates or. a-halogenated acetamide moieties. For conjugating chelating ligands to amine groups e.g. e-NH2-groups of lysine residues previous reduction of the peptide compounds is not required. The radiolabeled peptides are suitable for radio-diagnostic and radio-therapeutic applications.
The resulting radiolabeled peptides show unexpected advantages in animal experiments. For example, excretion of a labeled peptide, e.g. 99mTc-labeled AP39 (Seq. Id. No. 11) in nude mice occurs to 70% or more, e.g. 80.63% within 24 hours via the kidneys, whereas for L19 (Seq. Id. No. 1) labeled with 125I, excretion in nude mice occured only to 67.79% via the kidneys within 24 hours. The tumour to blood ratio of a labeled peptide, e.g. 99mTc-labeled AP39 is 5:1 or more, preferably 8:1 or more, e.g. about 10:1 after 5 hours, whereas for LI 9 labeled with 125I, this ratio is only about 3:1. This is an unexpected behaviour also compared to other scFv antibodies labeled with 99mTc which often show less favourable biodistribution characteristics. For example, Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868 - 872, 1 996, report a ""To-labeled scFv antibody that shows a tumour to blood ratio of only 4 : 1 after 24 hours, and a kidney accumulation of 9% after 24 hours, which is very high compared to the values of the peptides described in the present invention, e.g. 1.3% for 99mTc-labeled AP39 (see example 13 below).
Further, the in vivo stability of the labeled peptides of the invention, e.g. 99rTTc-labeled AP39 is much higher compared to the in vivo stability of L.1 9 labeled with 125I. The present inventors found that 2 hours after injection of
a peptide, e.g. ""To-labeled AP39 only 10% or less, e.g. 3% of radioactivity within the serum was due to a metabolite, whereas 2 hours after injection of L19 labeled with ~25\, 49% of the radioactivity in the serum was due to metabolites, which may be free iodine. The improved in vivo stability of the peptides, e.g. 99mTc-labeled AP39 is also reflected by a prolonged preservation of its binding ability to the target ED-B. The present inventors found that 2 hours after injection of the peptide, e.g. 99mTc-labeled AP39, 50% or more, e.g. 74% of radioactivity within the serum was able to bind ED-B, whereas 2 hours after injection of 1-125 labeled L1 9, only 27% of radioactivity within the serum could bind to ED-B. The compounds of this invention are also showing high tumour accumulation. Forexample, Tc-99m-AP39 and ln-111 -MX-DTPA-e-HN(Lys)-AP39 displayed high tumour accumulation of 10.7 (Tc-99m ) or 12.9 (In-111) % injected dose per gram (ID/g) at 1 hour post injection (p.i.). Thus, tumor uptake is significantly higher compared to other known ln-111 or Tc-99m labeled antibody fragments (e.g. Kobayashi et al., J. Nuc. Med., Vol. 41 (4), pp. 755 - 762, 2000; Verhaar et al., J. Nuc. Med., Vol. 37(5), pp. 868 - 872, 1996).
The compounds are suitable for diagnostic and therapeutic applications. They are preferably applied to the patient by parenteral administration, more preferably by intravenous injection. The human dose is preferably in the range of 0.1 to 1 mg per patient for radiodiagnostic applications, and 0.1 to 10O mg per patient for radiotherapeutic applications.
The methods for making and labeling the compounds of the present invention are more fully illustrated in the following examples. These examples are shown by way of illustration and not by way of limitation.
Experimentai
Example 1: Production of L19 derivatives
A recombinant antibody (scFv L19, short name L19) against the extra domain B (ED-B) of a splice variant of fibronectin formed the starting material. scFv L19 had been isolated by means of phage display selection from a synthetic human antibody repertoire (Neri et al., 1997, Nature Biotechnol. 15: 1271; Pini etal.,-1998, J. Biol. Chem. 273: 21769). This recombinant antibody fragment is in the form of a so-called single chain antibody fragment (scFv) and consists of a VH and VL region connected by a linker sequence (see Seq. Id. No. 1). This scFv L19 has exceptionally high affinity for ED B (Kd: 5.4 x 1Q-11 M).
Various derivatives of L19 were produced by genetic manipulation (see Fig. 1). To modify L19, the scFv encoding DNA was amplified by PCR (polymerase chain reaction) using primers which coded for the additional sequences, and cloned into expression vectors.
L19 derivatives:
L19: without additional terminal modifications
LI 9 His: C-terminal His6 domain (His tag), for Ni chelate
chrornatography and for binding radioisotopes
AP38: C-terminal GlyGlyGlyCys domain for binding (via
Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes)
AP39: C-terminal GlyGlyGlyCysAla domain for binding
(via Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes)

L19-GlyCysGlyCys: C-terminal GlyCysGlyCys domain for binding (via
Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes)
L19-GlyCysGlyCysAla: C-terminal GlyCysGlyCysAla domain for binding
(via Cys) substances which can be employed in therapy and diagnosis (e.g. radioisotopes)
Recombinant production of LI9 derivatives
The L19 derivatives described were produced in prokaryotic and eukaryotic expression systems.
a) LI 9 production in E. coli
The DMA sequences encoding various L19 derivatives (AP38, AP39, L19-GlyCysGlyCys, L19-GlyCysGlyCysAla, L19, L19His) were cloned into a prokaryotic expression vector (pDN5, Pini et al., 1997, J. Immunol, Methods 206: 171, Pini et al., 1998, J. Biol. Chem. 273: 21769; pET, Novageh) with IPTG-inducible promoter and ampicillin resistance marker. In order to make secretion of the recombinant protein into the periplasm possible, this vector was used to produce an expression cassette in which the N terminus of scFv is fused to a Pel B signal sequence. It was possible to establish stable producer strains by transforming E. coli (TG1, BL21DE3 and HB2151) with this expression vector, followed by ampicillin selection. To produce scFv, these strains were cultivated in the presence of 1 % glucose in the growth phase (37°C) in order to repress the promoter. Expression of scFv in the cultures was induced by adding IPTG and incubating at 30°C for up to 16h. Soluble and antigen-binding scFv material could be isolated from the complete extract of the E. coli strains, from the periplasm fraction or, which proved to be particularly efficient in relation to purification and yield, from the culture supernatant. Production

took place in shaken flasks and in fermenters with a culture volume of up to 10 litres.
b) Production of LI 9 derivatives in Pichia pastoris
L19His, AP38, AP39, L19-GlyCysGryCys and L1 9-GlyCysGlyCysAla-encoding DNA sequences were amplified by PCR and cloned into E. coli and into the expression vectors pPIC9K and pGAP (Invitrogen) for production in the yeast Pichia pastoris. For expression of heterologous genes, pPIC9K contains a methanol-inducible promoter (AOX1), and pGAP contains the constitutive promoter of the GAPDH enzyme. In addition, these vectors contain respectively a geneticin resistance gene and a zeocin resistance gene for selection/amplification of the foreign gene and a signal sequence (from yeast a factor) for expression and secretion of the recombinant product. The AP39 expression cassette used codes for a fusion protein (a factor signal + L19 derivatives) which contains for signal sequence elimination only a Kex2 cleavage site and not the other cleavage sites of natural a factor processing. Stable transfected PP clones were established by electroporation of the linearized vectors into Pichia pastoris strains (e.g. pPIC9K-AP39 into strain GS115, pGAP^-AP39 into strain X33) and subsequent geneticin or zeocin selection. It was possible to use these clones to produce the said L19 derivatives as soluble secretory protein. The clones were cultivated at 30°C in BMGY medium or basal mineral medium. With clones based on pPIC, methanol was added for promoter induction during the expression phase. The recombinant product had a correctly processed terminus and high antigen-binding activity. The yields which could be achieved (unpurified, bioactive product/litre of culture supernatant) were, depending on the culturing conditions and process control: e.g. pPIC9K-AP39/GS115 (shaken flask 5 mg/l, fermenter 10-15mg/l); pGAP-AP39/X33 (shaken flask 30-40 mg/l, fermenter 100 250 mg/l).
The L19 derivatives were purified from the Pichia pastoris or E, coli culture supernatant by use of affinity chromatography (rProtein A, Streamline Pharmacia or ED B antigen column) with subsequent size exclusion chromatography. The purified AP39 fraction, which was employed for further processing, had a homodimer structure (with subunits covalently linked for the most part) and high antigen-binding activity.
Example 2a
Synthesis of reduced AP38 [Reduced L19-(Gly)3-Cys-OH]
To a solution of 240 /yg (4.29 nmol) S-S-dimeric AP38 in 156 fj\ PBS {phosphate buffered saline)/! 0% glycerine were added 50//I TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na2HPO4, 0.1 M, pH = 7.4),. The reaction mixture was gently shaken for 1 h at room temperature. SH-tnonomeric AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S-S-dimeric AP38 to SH-rnonomeric AP38.
Yield: 79.4 /yg/220 jj\ PBS (33.1%).
Example 2b
Synthesis of Tc-99m-AP38 [Tc-99m-L19-(Gly)3-Cys-OH]
2,37 mg disodium-L-tartrate were placed in a vial followed by addition of 79.4 //g reduced AP38 in 220 //I PBS and the solution was diluted with 100 fj\ aqueous Na2HP04-buffer (1 M, pH = 10.5). 50 A/I Tc-99m generator
eluate (24 h) and 10//I SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 0.5 h at 37°C. Tc-99m-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 39.7%.
Radiochemical purity: 92.5% (SDS-PAGE).
Specific activity: 17.7 MBq/nmol.
Immunoreactivity: 88.7%
Example 3a
Synthesis of reduced AP39 [Reduced L19-(Gly)3-Cys-Ala-OH]
To a solution of 240//g (4.29 nmol) S-S-dimeric AP39 in 135/vl PBS/10% glycerine were added 50 //I TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na2HPO4, 0.1 M, pH = 7.4). The reaction mixture was gently shaken for 1 h-at room temperature. SH-monomeric AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S.-S-dimeric AP39 to SH-monomeric AP39.
Yield: 135.9 //g/1 80 u\ PBS (56.2%).
Example 3b
Synthesis of Tc-99m-AP39 [Tc-99m-L19-(GIy)3-Cys-Ala-OH]
4.2 mg disodium-L-tartrate were placed in a vial followed by addition of 135.9 fjg reduced AP39 in 180 //I PBS and the solution was diluted with 100 //I aqueous Na2HPO4-buffer (1 M, pH = 10.5). 100 jj\ Tc-99m generator eluate (24 h) and 10//I SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 0.5 h at 37°C. Tc-99m-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 50.1%.
Radiochemical purity: 91.5% (SDS-PAGE).
Specific activity: 21.4 MBq/nmol.
Irnmunoreactivity: 96.4%
Example 4
Synthesis of Re-188-AP38 [Re-188-L19-(Gly)3-Cys-OH]
2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 1 12>L/g reduced AP38 in 310/y| PBS and the solution was diluted with 100 /j\ aqueous Na2HPO4-buffer (1 M, pH = 10.5). 100//I Re-188 generator eluate and 50 jj\ SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 1.5 h at 37°C. Re-188-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 28.3%.
Radiochemical purity: Specific activity: Immune-reactivity:91.1% -(SOS-PAGE).
15.3 MBq/nmol. 89.9%

Example 5
Synthesis of Re-188-AP39 [Re-188-L19-(Gly)3-Cys-Ala-OH]
2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 1 12/yg reduced AP39 in 303//I PBS and the solution was diluted with 100 //I aqueous Na2HPO4-buffer (1 M, pH = 10.5). 100 fj\ Re-188 generator eluate and 50/vl SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaken for 1.5 h at 37°C. Re-188-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).

Radiochemical yield: Radiochemical purity: Specific activity: Immunoreactivity:

33.5%.
92.3% .(SOS-PAGE).
18.5 MBq/nmol.
92.5%

Example 6a
Synthesis of reduced L19-Gly-Cys-Gly-Cys-OH
To a solution of 240 //g (4.29 nmol) S-S-dimeric L19-Gly-Cys-Gly-Cys-OH in 160//I PBS/10% glycerine were added 75 //I TCEP-solution (14.34 rng TCEP x HCI/5 ml aqueous Na2HP04, 0.1 M, pH - 7.4). The reaction
mixture was gently shaken for 1 h at room temperature. SH-monomeric L1 9-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S-S-dimeric L19-Giy-Cys-Gly-Cys-OH to SH-monomeric L1 9-Gly-Cys-Gly-Cys-OH.
Yield: 80.4//g/210//I PBS (33.5%).
Example 6b
Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-OH
2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 80.4/yg reduced L19-Gly-Cys-Gly-Cys-OH in 210/yl PBS and the solution was diluted with 100//I aqueous Na2HPO4-buffer (1 M, pH = 10.5). 50/vl Tc-99m generator eluate (24 h) and 10 fj\ SnCI2-solution (5 mg SnCI2/l rnl 0.1 M HCI) were added. The reaction mixture was shaked for 0.5 h at 37°C. Tc-99m-labeled L.19-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 37.7%.
Radiochemical purity: 91.5% (SDS-PAGE).
Specific activity: 19.7 MBq/nmol.
Irnmunoreactivity: 89.7%
Example 7a
Synthesis of reduced L19-Gly-Cys-Gly-Cys-AIa-OH
To a solution of 240//g (4.29 nmol) S-S-dimeric L19-Gly-Cys-Gly-Cys-Ala-OH in 155 //I PBS/10% glycerine were added 75//I TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na2HPO4, 0.1 M, pH = 7.4). The reaction mixture was gently snaked for 1 h at room temperature. SH-monomeric L1 9-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product proofed the quantitative transformation of S-S-dimeric L19-Gly-Cys-Gly-Cys-Ala-OH to SH-monomeric L19-Gly-Cys-Gly-Cys-Ala-OH.
Yield: 81.2//g/215//I PBS (33.8%).
Example 7b
Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-Ala-OH
2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 81.2 jjg reduced L19-Gly-Cys-Gly-Cys-Ala-OH in 215 //I PBS and the solution was diluted with 100 jj\ aqueous Na2HPO4-buffer (1 M, pH = 10.5). 50/ylTc-99m generator eluate (24 h) and 10//I SnCI2-solution (5 mg SnCI2/1 ml 0.1 M HCI) were added. The reaction mixture was shaked for 0.5 h at 37°C. Tc-99m-labeled L19-Gly-Cys-Gly-Cys-Ala-OH was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 35.6%. Radiochernical purity: 93.5% (SDS-PAGE).

Specific activity: 19.1 MBq/nmol.
Immunoreactivity: 88.7%
Example 8a
Synthesis of reduced AP39 for specific conjugation of EDTA, CDTA, TETA, DTPA, TTHA, HBED, DOTA, NOTA, and DO3A type chelators to the Cysteine-SH group
50//I TCEP-solution (14.34mg TCEPxHCI/5ml aqueous Na2HPO4/ 0.1M, pH = 7.4) were added to a solution of 400/yg (7.1 nmol) AP39 in 450//I PBS. The reaction mixture was gently shaken for 1h at 37 °C. Reduced AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: sodium acetate buffer, 0.1M, pH 5.0). SDS-PAGE analysis of the isolated product proofed the complete? transformation of AP39 into reduced AP39.
Yield: 140//g/200//l (35%). Example 8b
Synthesis of MX-DTPA-Maleimide (1,4,7-triaza-2-{N-maleimido ethylene/?-amino)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl 6-methyl heptane)
512 mg (1 mmol) of {[3-(4-Arnino-phenyl)-2-(bis-carboxymethyl-amino)-propyl]-[2-(bis-carboxymethyl-amino)-propyl]-amino}-acetic acid (Macrocyclics Inc. Dallas, TX, U.S.A.) and 707 mg (7 mmol) triethylarnine were dissolved in 3 ml dry DMF. 400 mg (1,5 mmol) of 3-(2,5-Dioxo-2,5-d)hydro-pyrrol-1-yl)-propionic acid 2,5-dioxo-pyrrolidin-1-yl ester (Aldrich)

in 1 ml dry DMF were added dropwisely. The solution was stirred for 5 h at 50° C. 30 ml of diethylether were added slowly. The reaktion mixture was stirred for further 30 min. The principate was collected by filtering. The crude product was purified by RP-HPLC (acetonitrile- : water- : trifluoracetic acid / 3 : 96,9 : 0,1 -*• 99,9 : 0 : 0,1). Yield: 61% (405 mg, 0,61 mmol). MS-ESI: 664 = M+ +1.
Example 8c
Synthesis of ln-111-MX-DTPA-MaIeirnide-S{Cys)-AP39-R
(R = reduced)
140/yg (5 nmol) AP39-R in 200 /j\ of sodium acetate buffer (0.1M, pH 5) were reacted with 50/c/l of dissolved 1,4,7-triaza-2-(N-maleimido ethylene p-amino) benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl6-methyl heptane (0,25mg DTPA-Maleimide in 500//I sodium acetate buffer 0.1M pH 5) for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h with 200rnl of sodium acetate buffer (0.1 M, pH 6) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.).
80 p\ [ln-1 11 ]lnCI3 solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37 °C for 30 min. ln-111 labeled DTPA-Maleimide-S(Cys)-AP39-R was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 54 %.
Radiochemical purity: 94 % (SDS-PAGE).
Specific activity: 6.2 MBq/nmol.
Immunoreactivity: 86 %

Example 9
Synthesis of ln-111-MX-DTPA-e-HN(Lys)-AP3£
200//g (3.6 nmol) non-reduced AP39 in 111 /j\ PBS were diluted with 300 fj\ of sodium borate buffer (0.1M, pH 8.5} and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 50 //I of 1,4,7~triaza-2-(p-isothiocyanato) benzyl- 1,7-bis(carboxyrriethyl)-4-carboxymethyl-6-rnethyl heptane (MX-DTPA) solution (0.33 mg MX-DTPA dissolved in 500 //I of sodium borate buffer, 0.1M, pH 8.5) were added and the reaction mixture was heated for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h and 1 x 1 7 h (over night) with 200 ml of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.).
80 A/I [ln-1 11]lnCI3 solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37°G for 30 min. ln-111 labeled MX-DTPA-e-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 70 %.
Radiochemical purity: 85 % (SDS-PAGE).
Specific activity: 7.6 MBq/nmol.
Irnmunoreactivity: 74 %
Example 10
Synthesis of ln-111 -DOTA-C-Benzyl-p-NCS -e-HN(Lys)-AP39
200//g (3.6 nmol) non-reduced AP39 in 114//I PBS were diluted with 300 fj\ of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc.,. Rockford, IL, U.S.A.). 50//I of 1,4,7,1O-tetraaza-2-i isothiocyanato)benzyl cyclododecane-1,4,7,10-tetraacetic acid (benzyl-p-SCN-DOTA, Macrocyclics Inc., Dallas TX, U.S.A.) solution (1.5 mg benzyl-p-SCN-DOTA dissolved in 5 ml of sodium borate buffer, 0.1M, pH 8.5) were added to the solution and the reaction mixture was heated for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h and 1 x 17 h (over night) with 200 ml of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.).
80 fj\ [ln-111 ]lnCI3 solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37°C for 30 min. ln-111 labeled DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 74 %.
Radiochemical purity: 94 % (SDS-PAGE).
Specific activity: 12.3 MIBq/nmol.
Immunoreactivity: 73 %
Example 11
Synthesis of Y-88-MX-DTPA-e-HN(Lys)-AP39
200 jug (3.6 nmol) non-reduced AP39 in 115//I PBS were diluted with 300 //I of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,OOO MWCO (Pierce Inc., Rockford, IL, U.S.A.). 50 //I of MX-DTPA solution (0.33 mg MX-DTPA dissolved in 500/vl of sodium borate buffer, 0.1M, pH 8.5) were added and the reaction mixture was heated for 3 h at 37 ° C. The reaction mixture was dialyzed 2 x 1 h and 1 x 17 h (over night) with 200 rnl of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.).
100 //I [Y-88]YCI3 solution (HCI, 1N, 75 MBq, Oak Ridge National Lab.) were added and the reaction mixture was heated at 37°C for 30 min. Y-88 labeled MX-DTPA-«r-HN(LyshAP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).
Radiochemical yield: 65 %.
Radiochemical purity: 93 % (SDS-PAGE).
Specific activity: 10.2 MBq/nmol.
Irnmunoreactivity: 72 %
Example 12
Synthesis of Lu-177 -DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP3
200/vg (3.6 nmol) non-reduced AP39 in 110//I PBS were diluted with 300 //I of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200ml of sodium borate buffer (0.1M, pH 8.5) employing a Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.). 50//I of benzyl-p-SCN-DOTA solution {1.5 mg dissolved in 5 ml of sodium borate buffer, 0.1M, pH 8.5) were added and the reaction mixture was heated for 3 h at 37 °C. The reaction mixture was dialyzed 2 x 1 h and 1 x 1 7 h (over night) with 200 ml of sodium acetate buffer (0.1M, pH 6.0) each, employing the Slide-A-Lyzer 10,000 MWCO (Pierce Inc., Rockford, IL, U.S.A.).
200//I [Lu-177]LuCI3 solution (HCI, 1N, 80MBq, NRH-Petten, Netherlands) were added and the reaction mixture was heated at 37 °C for 30 min. Lu-177 labeled DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP39 was purified by gel-chromatography using.a NAP-5-column (Amersham, Eluent: PBS).
Radiochemical yield: 74 %.
Radiochemical purity: 95 % (SDS-PAGE).
Specific activity: 19 MBq/nmol.
Immunoreactivity: 71 %

Example 13
Organ distribution and excretion of Tc-99m-AP39, expressed in Pichia pastoris, after a single i.v. injection into tumour-bearing nude mice
The substance of the invention is injected intravenously in a dose of about 74 kBq into F9 (teratocarcinoma)-bearing animals (bodyweightabout25 g). The radioactivity concentration in various organs, and the radioactivity in the excreta are measured using a y counter at various times after administration of the substance. In addition, the tumour to blood ratio is found at various times on the basis of the concentration of the substance of the invention in tumour and blood,
The biodistribution of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice {mean ± SD, n = 3) is shown in Table 2:

(Table Removed)
Table 2
The excretion of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Table 3:

(Table Removed)Table 3
The tumour to blood ratio of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Fig. 2.
The results of this investigation show the excellent potential of the substance of the invention for accumulation in solid tumours with, at the same time, excellent excretion.
Example 14
Organ distribution of ln-111-MX-DTPA- The substance of the invention is injected intravenously in a dose of about 48 kBq into F9 (teratocarcinoma)-bearing animals (body weight about 25 g). The radioactivity concentration in various organs, and the radioactivity in the excreta are measured using a y counter at various times after administration of the substance,
The biodistribution of ln-111-MX-DTPA-e-HN(Lys)-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Table 4:

(Table Removed)Table 4

The tumour to blood ratio of ln-111-MX-DT.PA-e-HN(l_ys)-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean ± SD, n = 3) is shown in Table 5.

(Table Removed)Table 5
The results of this investigation show the excellent potential of the substance of the invention for accumulation in solid tumours paired with excellent biodistribution and tumor to blood ratio.
Example 15
Imaging of Tc-99m-AP39, expressed in Pichia pastoris, after a single i.v. injection into tumour-bearing nude mice
The substance of the invention is injected intravenously in a dose of about 9.25 MBq into F9 (teratocarcinoma)-bearing animals {bodyweight about 25 g). Gamma-camera imaging is carried out at various times after administration of the substance.
Planar scintigraphy of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice is shown in Figures 3 and 4. Fig. 3 shows the scintigram 5 hours after injection of the substance, and Fig. 4 shows the scintigram 24 hours after injection of the substance.
The result of this investigation shows the excellent potential of the substance of the invention for imaging solid tumours.

WE CLAIM:
1. A compound that effectively binds to radioisotopes and to extracellular ED-B domain of fibronectin for use in diagnosis and treatment of tumor comprising:
a peptide derived from recombinant ScFv antibody L19 according to SEQ ID NO: 1 comprising:
k) an antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity-determining regions HCDR3 (PFPYFDY) and/or LCDR3 (CQQTGRIPPT); or
l) an antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity-determining regions HCDR1 (SFSMS), HCDR2 (SISGSSGTTYYADSVKG), HCDR3 (PFPYFDY), LCDR1 (RASQSVSSSFLA), LCDR2 (YASSRAT) and LCDR3 (CQQTGRIPPT); or
m) a sequence according to Seq. Id. No. 1 (L19) and

u) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2), wherein Xaa1, Xaa2 and Xaa3 each independently represent any naturally occuring amino acid or
v) an amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4(Seq. Id. No. 3), wherein Xaa1, Xaa2, Xaa3, and Xaa4 each independently represent any naturally occuring amino acid or
bc) an amino acid sequence (His)n (Seq. Id. No. 4), wherein n stands for an integer from 4 to 6,
wherein the C-terminus of aa), ab) or ac) is bound to the N-terminus of one of the sequences Seq. Id. No. 2, Seq. Id. No. 3 or Seq. Id. No. 4 via a peptide bond.
2. The compound as claimed in claim 1, wherein the amino acid sequence Xaa1-Xaa2-Xaa3-Cys (Seq. Id. No. 2) is the sequence Gly-Gly-Gly-Cys (Seq. Id. No. 5) or Gly-Cys-Gly-Cys (Seq. Id. No. 6).
3. The compound as claimed in claim 1, wherein the amino acid sequence Xaa1-Xaa2-Xaa3-Cys-Xaa4 (Seq. Id. No. 3) is the sequence Gly-Gly-Gly-Cys-Ala (Seq. Id. No. 7) or Gly-Cys-Gly Cys-Ala (Seq. Id. No. 8).
4. The compound as claimed in claim 1, wherein n in the amino acid sequence (His)n (Seq. Id. No. 4) is 6.
5. The compound as claimed in any one of claims 1 to 4 which is optionally conjugated to a radioisotope.
6. The compound as claimed in claim 6 which is conjugated to a radioisotope selected from a radioisotope of Technetium, such as 94mTc, 99mTc, Rhenium, such as I86Re, 188Re, or other isotopes, such as 203Pb, 67Ga, 68Ga, 43Sc, ^Sc, 47Sc, 110mIn, 111In, 97Ru, 62Cu, 64Cu, 67Cu, 68Cu, 86Y, 88Y, 90Y, 121Sn, 161Tb, 153Sm, 166Ho, 105Rh, 177Lu, 72As and 18F.
7. A compound as claimed in claim 6, wherein the radioisotope is 99mTc or 188Re.
8. The compound as claimed in any one of claims 1 to 7, wherein the peptide is in reduced form.

Documents:

1523-DELNP-2004-Abstract-(18-05-2010).pdf

1523-DELNP-2004-Abstract-(19-05-2010).pdf

1523-DELNP-2004-Abstract-(27-02-2009).pdf

1523-delnp-2004-abstract.pdf

1523-DELNP-2004-Claims-(18-05-2010).pdf

1523-DELNP-2004-Claims-(19-05-2010).pdf

1523-DELNP-2004-Claims-(27-02-2009).pdf

1523-delnp-2004-claims.pdf

1523-delnp-2004-Correspondence Others-(21-07-2011).pdf

1523-DELNP-2004-Correspondence-Others (15-10-2009).pdf

1523-DELNP-2004-Correspondence-Others-(06-02-2009).pdf

1523-DELNP-2004-Correspondence-Others-(18-05-2010).pdf

1523-DELNP-2004-Correspondence-Others-(19-05-2010).pdf

1523-DELNP-2004-Correspondence-Others-(27-02-2009).pdf

1523-DELNP-2004-Correspondence-Others-(30-10-2008).pdf

1523-delnp-2004-correspondence-others.pdf

1523-DELNP-2004-Description (Complete)-(18-05-2010).pdf

1523-DELNP-2004-Description (Complete)-(27-02-2009).pdf

1523-delnp-2004-description (complete).pdf

1523-DELNP-2004-Drawings-(27-02-2009).pdf

1523-delnp-2004-drawings.pdf

1523-DELNP-2004-Form-1-(18-05-2010).pdf

1523-DELNP-2004-Form-1-(27-02-2009).pdf

1523-delnp-2004-form-1.pdf

1523-delnp-2004-form-13-(30-10-2008).pdf

1523-delnp-2004-form-18.pdf

1523-DELNP-2004-Form-2-(18-05-2010).pdf

1523-DELNP-2004-Form-2-(27-02-2009).pdf

1523-delnp-2004-form-2.pdf

1523-delnp-2004-form-3.pdf

1523-delnp-2004-form-5.pdf

1523-DELNP-2004-GPA-(27-02-2009).pdf

1523-DELNP-2004-Others-Document-(30-10-2008).pdf

1523-delnp-2004-pa.pdf

1523-delnp-2004-pct-101.pdf

1523-delnp-2004-pct-210.pdf

1523-delnp-2004-pct-304.pdf

1523-delnp-2004-pct-306.pdf

1523-delnp-2004-pct-409.pdf

1523-delnp-2004-pct-416.pdf

1523-DELNP-2004-Petition-137-(27-02-2009).pdf

1523-DELNP-2004-Petition-138-(27-02-2009).pdf

1523-delnp-2004-petition-138.pdf

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Patent Number

240962

Indian Patent Application Number

1523/DELNP/2004

PG Journal Number

25/2010

Publication Date

18-Jun-2010

Grant Date

10-Jun-2010

Date of Filing

02-Jun-2004

Name of Patentee

SCHERING AKTIENGESELLSCHAFT

Applicant Address

MULLERSTRASSE 178, 13353 BERLIN, GERMANY.

Inventors:

#	Inventor's Name	Inventor's Address
1	CHRISTOPH-STEPHAN HILGER	LANGENAUER WEG 24, 13503 BERLIN, GERMANY.
2	DIETMAR BERNDORFF	EICHENALLEE 79A, 16540 HOHEN NEUENDORF, GERMANY.
3	LUDGER DINKELBORG	ORTWINSTRASSE 7, 13465 NEUENDORF, GERMANY.
4	DIETER MOOSMAYER	SCHERING AKTIENGESELLSCHAFT, MULLERSTRASSE 178, 13353 BERLIN, GERMANY.
5	GIOVANNI NERI	PHILOGEN S.R.L. LALIZZA 7, I-52100 SIENA, ITALY

PCT International Classification Number

C07K 19/00

PCT International Application Number

PCT/EP03/00009

PCT International Filing date

2003-01-02

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/358,702	2002-02-25	U.S.A.