KR20040073540A - Conjugates comprising an antibody specific for the ed-b domain of fibronectin and their use for the detection and treatment of tumours - Google Patents

Abstract

The present invention relates to methods for diagnosis and treatment of tumours, using peptides for binding radionuclides. The peptides comprise an antigen- binding site for the extra domain B (ED-B) of fibronectin.

Description

CONJUGATES COMPRISING AN ANTIBODY SPECIFIC FOR THE ED-B DOMAIN OF FIBRONECTIN AND THEIR USE FOR THE DETECTION AND TREATMENT OF TUMOURS}

Tumors cannot be heavier than a certain weight without the creation of new blood vessels (angiogenesis), and the relationship between microtubule density and tumor permeability has been reported for many tumors (Folkman (1995), Nature Med., 1, 27-31). Moreover, angiogenesis leads to loss of vision associated with most eye diseases (Lee et al., Surv. Ophthalmol. 43, 245-269 (1998); Friedlander, M. et al., Proc. Natl. Acad Sci. USA 93, 9764-9769 (1996). Molecules capable of selectively targeting markers of angiogenesis will provide clinical opportunities for the diagnosis and treatment of tumors and other diseases characterized by vascular proliferation, such as diabetic retinopathy and age-related macular degeneration. Angiogenic markers will be readily accessible to specific conjugates expressed in the majority of aggressive solid tumors associated with tumor vessels and thus injected intravenously (Pasqualini et al., (1997), Nature Biotechnol., 15, 542-546; Neri et al. (1997), Nature Biotechnol., 15, 1271-1275). Target obstruction of neovascular structures can lead to tumor 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, the same 91 amino acid sequence in mice, rats, and humans, inserted by alternative splicing into the fibronectin molecule, is specifically accumulated around the neovascular structure (Castellani et al. (1994), Int. J. Cancer 59, 612-618). Indeed, fluorescence technology recently revealed that anti-ED-B single-chain Fv antibody fragments (scFv) selectively accumulate around tumor vessels in tumor-bearing mice and that antibody affinity presumably determines target performance (Neri et al. ( 1997), Nature Biotechnol., 15, 1271-1275; WO 97/45544).

In addition, antibodies and antibody fragments as well as radioisotope labeled derivatives thereof that specifically bind to the ED-B domain of fibronectin with dissociation constants of up to nanomolar are described in WO 99/58570. Biodistribution of ¹²⁵ I-labeled antibody fragments called L19, one of the high-affinity human antibody fragments, has already been studied in tumor-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192-198). Radioisotope labeled conjugates comprising L19-antibodies and their use for the detection and treatment of angiogenesis are disclosed in WO 01/62800.

Recombinant preparation of functionalized single-chain Fv antibody fragments that bind to the ED-B domain of the B- homolog of fibronectin of Pichia pastoris has already been described (Marty et al., Protein Expression and Purification 21, 156). -164 (2001)).

In addition, radioisotope labels via C-terminal cysteine peptides with scFv antibody fragments and ^99m Tc are described by George et al., Proc. Natl. Acad. Sci. USA, Vol. 92 pp. 8358-8362, 1995, and Verhaar et al., J. Nuc. Med., Vol. 37 (5), pp. 868-872, 1996.

However, since radionuclides are particularly suitable for radiopharmaceuticals, there is still a clinical need for the provision of antibody fragments with improved pharmacokinetic properties and which can be easily labeled with radioisotopes such as technetium or rhenium.

<Object of invention>

It is therefore an object of the present invention to provide antibody fragments which have improved pharmacokinetic properties, in particular target specificity and / or in vivo stability, and which can readily bind to radioisotopes such as technetium or rhenium.

Summary of the Invention

The present invention

aa) sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and / or LCDR3 shown in Table 1 or having the same function as the peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions, and / or substitutions of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region;

ab) the peptide according to SEQ ID NO: 1 or the sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 3 amino acids or less in HCDR1 region, 8 amino acids or less in HCDR2 region, 5 amino acids or less in HCDR3 region, 6 amino acids or less in LCDR1 region, 4 amino acids or less in LCDR2 region, Variants thereof which are deletions, insertions and / or substitutions of up to 6 amino acids in the LCDR3 region;

ac) a variant of SEQ ID NO: 1 that is a deletion, insertion, and / or substitution of up to 30 amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and

ba) the amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys (SEQ ID NO: 2), wherein Xaa ₁ , Xaa ₂ , and Xaa ₃ each independently represent any naturally occurring amino acid; or

bb) Amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys-Xaa ₄ (SEQ ID NO: 3), wherein Xaa ₁ , Xaa ₂ , Xaa ₃ , and Xaa ₄ each independently represent any naturally occurring amino acid or

bc) amino acid sequence (His) _n (SEQ ID NO: 4), where n represents an integer from 4 to 6

Wherein the C-terminus of aa), ab) or ac) comprises a peptide linked to the N-terminus of one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 via peptide bonds Describe the compound.

The compound is preferably a single chain antibody fragment, in particular an scFv fragment. In addition, the compound may be a radioisotope such as technetium, for example ^94m Tc, ^99m Tc, rhenium, for example ¹⁸⁶ Re, ¹⁸⁸ Re, or other isotope, for example ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga , ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m In, ¹¹¹ In, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁵³ Sm, ¹⁶⁶ It is preferably conjugated to radioisotopes of Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As and ¹⁸ F.

The present invention also describes pharmaceutical compositions comprising a physiologically acceptable adjuvant, diluent and / or carrier with the compound as an active agent.

The invention also

aa) sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and / or LCDR3 shown in Table 1 or having the same function as the peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions, and / or substitutions of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region;

ab) the peptide according to SEQ ID NO: 1 or the sequence of the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 3 amino acids or less in HCDR1 region, 8 amino acids or less in HCDR2 region, 5 amino acids or less in HCDR3 region, 6 amino acids or less in LCDR1 region, 4 amino acids or less in LCDR2 region, Variants thereof which are deletions, insertions and / or substitutions of up to 6 amino acids in the LCDR3 region;

ac) a variant of SEQ ID NO: 1 that is a deletion, insertion, and / or substitution of up to 30 amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and

ba) the amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys (SEQ ID NO: 2), wherein Xaa ₁ , Xaa ₂ , and Xaa ₃ each independently represent any naturally occurring amino acid; or

bb) Amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys-Xaa ₄ (SEQ ID NO: 3), wherein Xaa ₁ , Xaa ₂ , Xaa ₃ , and Xaa ₄ each independently represent any naturally occurring amino acid or

bc) amino acid sequence (His) _n (SEQ ID NO: 4), where n represents an integer from 4 to 6

Wherein the C-terminus of aa), ab), or ac) is linked to the N-terminus of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 via a peptide bond Describe the use of radioactive isotopes of elements such as technetium or rhenium.

Antibody fragment L19 is defined by the following sequence (SEQ ID NO: 1):

(VH)

EVQLLESGGG LVQPGGSLRL SCAASGFTFS

SFSMSWVRQA PGKGLEWVSS ISGSSGTTYY

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF PYFDYWGQGT LVTVSS

(Linker)

GDGSSGGSGG ASTG

(VL)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS

SSFLAWYQQK PGQAPRLLIY YASSRATGIP

DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ

QTGRIPPTFG QGTKVEIK

Deletions, insertions, and / or substitutions of up to 30 amino acids may be performed at 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 of SEQ ID NO: 1 , 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids deleted, inserted and / or substituted. However, variants that are peptides claimed within the complementarity-determining region (CDR), eg, the peptide of SEQ ID NO: 1, deletion, insertion and / or substitution of amino acids (aa), exceed the maximum variation defined in Table 1 below. (HCDR: CDR of heavy chain; LCDR: CDR of light chain).

area CDR length (aa) order Maximum (preferred) variation in sequence position HCDR1 5 SFSMS 3 (2,1) HCDR2 17 SISGSSGTTYYADSVKG 8 (7,6,5,4,3,2,1) HCDR3 7 PFPYFDY 5 (4,3,2,1) LCDR1 12 RASQSVSSSFLA 6 (5,4,3,2,1) LCDR2 7 YASSRAT 4 (3,2,1) LCDR3 10 CQQTGRIPPT 6 (5,4,3,2,1)

CDRs are described in 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 variant of SEQ ID NO: 1 which is a sequence according to SEQ ID NO: 1 (L19) or a deletion, insertion and / or substitution of up to 20 amino acids.

Peptides comprising the mutations in the CDR sequences shown in Table 1 and in particular the mutations in SEQ ID NO: 1 which are deletions, insertions and / or substitutions having the same function as the peptides according to SEQ ID NO: 1 are measured by BIAcore. It is defined as a peptide that binds to the ED-B domain of fibronectin with a dissociation constant K _d in the sub-molar range (ie lower than 10 ⁻⁹ ) (see WO99 / 58570, Example 2 and Table 2).

Preferred amino acid sequences Xaa ₁ -Xaa ₂ -Xaa ₃ -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 Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys-Xaa ₄ (SEQ ID NO: 3) include 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 the amino acid sequence (His) _n (SEQ ID NO: 4), compounds in which n is an integer 6 are preferred.

Preferred radioisotopes of technetium or rhenium are isotopes ^94m Tc, ^99m Tc, ¹⁸⁶ Re and ¹⁸⁸ Re. Most preferred is the radioisotope ^99m Tc.

The present invention relates to novel methods for the diagnosis and treatment of tumors using novel peptides that bind to radionuclides.

Single-chain antibody fragment L19 (SEQ ID NO: 1) was previously labeled ¹²⁵ I to examine the biodistribution of this compound in tumor-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p) 192-198). The results show that selective targets of tumor vessels in vivo can be achieved. However, it was surprisingly found that the pharmacokinetic properties of single chain antibody fragment L19 can be significantly improved when conjugated with peptides ba), bb) or bc) and labeled with the radioisotopes of technetium or rhenium. Isotope ^99m Tc has radiochemical properties (easily obtainable via ⁹⁹ Mo / ^99m Tc generator, emits a single gamma-photon of 140KeV, has a high photon flux, decays in 6 hours half-life) and cost- Because of its utility, it is the radioisotope label of choice for routine clinical SPECT. For therapeutic applications, labels with chemically homologous isotopes ¹⁸⁶ Re and ¹⁸⁸ Re are particularly preferred (Hsieh, BT, et al., Nucl. Med. Biol., 1999, 26 (8), 967-972; 973 -976, Zamora, PO, et al., Anticancer Res., 1997, 17 (3B), 1803-1838).

Peptides of the invention are derivatives of the recombinant scFv antibody L19 (SEQ ID NO: 1) for the extracellular ED-B domain of fibronectin and were prepared via genetic engineering according to FIG. The following peptides were prepared:

L19 (SEQ ID NO: 1)

L19His:

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS

LSPGERATLS

151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR

FSGSGSGTDF

201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKAAAL

EHHHHHH

(SEQ ID NO: 9)

AP38:

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS

LSPGERATLS

151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR

FSGSGSGTDF

201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC

(SEQ ID NO: 10)

AP39:

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS

LSPGERATLS

151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR

FSGSGSGTDF

201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC A

(SEQ ID NO: 11)

L19-GlyCysGlyCys:

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS

LSPGERATLS

151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR

FSGSGSGTDF

201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC

(SEQ ID NO: 12)

L19-GlyCysGlyCysAla:

1 EVQLLESGGG LVQPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASEI VLTQSPGTLS

LSPGERATLS

151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR

FSGSGSGTDF

201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGCGC A

(SEQ ID NO: 13)

Preparation of the peptides is described in detail in the Examples below (see "Examples").

Antibody fragment L19 is E. coli. Originally made by expression in E. coli (see WO 99/58570). However, for large scale preparation of scFv antibody fragments, this expression system has been found to be unsatisfactory. Another expression system, the yeast expression system, in particular the Pichia pastoris expression system, was tested. Applicants are generally capable of expressing highly fragmented antibody fragments such as yeast, eg, Pichia pastoris , for example fragment AP39, but 250 mg antibody per liter culture required for economic preparation of biopharmaceuticals. It has been found that high yield expression up to fragments can always be obtained by expression vectors (eg pGAP) and not by methanol inducible vectors (eg pPIC9K). An additional advantage of this always expression system is the simplified and strong fermentation process compared to inducible yeast expression. Unexpectedly, Applicants have found that proper signal sequence processing of antibody fragments, eg, fragment AP39, is observed only when an expression cassette in which the N-terminus of the fragment is directly fused to the Kex2-cleavage site of the alpha-signal sequence is used.

Peptides are suitable for diagnostic and therapeutic uses, in particular for the diagnosis and treatment of invasive tumors and tumor metastases. Preferred diagnostic applications are SPECT (Single Photon Emission Computed Tomography) and PET (Positron Emission Tomography).

The peptides are particularly suitable for the labeling of radioisotopes of these radioisotopes, such as technetium and rhenium, preferably radionuclides ^94m Tc, ^99m Tc, ¹⁸⁶ Re, and ¹⁸⁸ Re. For labeling of the peptide, the peptide is first reduced with a suitable reducing agent such as tin chloride or tris (2-carboxyethyl) phosphine (TCEP). The resulting reduced peptides present SH-groups which can be reacted with ^99m Tc generator eluents or ¹⁸⁸ Re generator eluents and tin chlorides to form compounds of the invention (see examples below for details). Indirect labeling is carried out by pre-conjugation of the chelating ligand and subsequent complexation of radioisotopes such as indium, yttrium, lanthanides and the like. The chelating ligand is preferably ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), cyclohexyl 1,2-diaminetetraacetic acid (CDTA), ethylene glycol-O, 0'-bis (2-amino Ethyl) -N, N, N ', N'-diacetic 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, N ', N ", N"'-tetraacetic acid (TETA) is derived from the amine or thiol group of the peptide compound. Chelating ligands have suitable coupling groups such as active esters, maleimides, thiocarbamates or α-halogenated acetamide residues. In order to conjugate the chelating ligand to an amine group, for example the ε-NH ₂ -group of the lysine residue, no prior reduction of the peptide compound is required. Radioisotope labeled peptides are suitable for radio-diagnostic and radio-therapeutic applications.

The resulting radioisotope labeled peptides show unexpected advantages in animal experiments. For example, the release of labeled peptides, such as ^99m Tc-labeled AP39 (SEQ ID NO: 11), in nude mice occurs more than 70%, eg 80.63%, within 24 hours through the kidney, while ¹²⁵ I Labeled L19 (SEQ ID NO: 1) produced only 67.79% of the excretion in nude mice within 24 hours through the kidney. Tumor-to-blood ratios of labeled peptides such as ^99m Tc-labeled AP39 are at least 5: 1 after 5 hours, preferably at least 8: 1, for example about 10: 1, whereas in L19 labeled ¹²⁵ I This ratio is only about 3: 1. This is also an unexpected response compared to other ^99m Tc-labeled scFv antibodies, which often show less favorable biodistribution characteristics. See, eg, Verhaar et al., J. Nuc. Med., Vol. 37 (5), pp. 868-872, 1996 are very high compared to the value of the peptides described herein, for example, 1.3% for ^99m Tc-labeled AP39, a tumor to blood ratio of only 4: 1 after 24 hours, and after 24 hours. Report ^99m Tc-labeled scFv antibodies showing 9% kidney accumulation (see Example 13 below).

Moreover, the in vivo stability of the labeled peptides of the invention, for example ^99m Tc-labeled AP39, is very high compared to the in vivo stability of L19 labeled ¹²⁵ I. We found that only 2% or less, eg 3%, of radioactivity in serum was due to metabolites 2 hours after injection of peptides such as ^99m Tc-labeled AP39 2 while injection of L19 labeled ¹²⁵ I 2 After time it was found that 49% of the radioactivity in the serum was due to a metabolite that could be free iodine. Improved in vivo stability of peptides such as ^99m Tc-labeled AP39 also reflects continued preservation of the binding capacity to target ED-B. The inventors of the labeled L19, ¹²⁵ I, while the peptide, for example, ^99m Tc- after injection of the labeled AP39 2 sigan serum example within 50% or more, for example, 74% of radioactivity capable of binding to the ED-B Two hours after injection, only 27% of the radioactivity in the serum was found to bind to ED-B. The compounds of the present invention also show a high degree of tumor accumulation. For example, Tc-99m-AP39 and In-111-MX-DTPA-ε-HN (Lys) -AP39 are 10.7 (Tc-99m) or 12.9 (In-111) at 1 hour post injection (pi). A) showed a high tumor accumulation of per injection per gram (ID / g). Therefore, tumor uptake is significantly higher than other known In-111 or Tc-99m labeled antibody fragments (eg, 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 use in diagnosis and treatment. They are preferably applied to the patient by parenteral administration, more preferably by intravenous injection. The human dosage is preferably in the range of 0.1 to 1 mg per patient for radiodiagnostic use and in the range of 0.1 to 100 mg per patient for radiotherapy use.

The preparation and labeling methods of the compounds of the present invention are more fully illustrated in the following examples. These embodiments have been shown for purposes of illustration and not limitation.

Example 1: Preparation of L19 Derivatives

Recombinant antibody against the extracellular domain B (ED-B) of the splice variant of fibronectin (scFv L19, abbreviated L19) formed the starting material. scFv L19 was isolated from synthetic human antibody repertoire by phage display screening (Neri et al., 1997, Nature Biotechnol. 15: 1271; Pini et al., 1998, J. Biol. Chem. 273: 21769). This recombinant antibody fragment is in the form of so-called single chain antibody fragment (scFv) and consists of the VH and VL regions linked by linker sequences (see SEQ ID NO: 1). scFv L19 has an extremely high affinity for ED B (K _d : 5.4 × ^{10 −11} M).

Various derivatives of L19 were prepared by genetic manipulation (see FIG. 1). To modify L19, scFv coding DNA was amplified by PCR (polymerase chain reaction) using primers encoding additional sequences and cloned into expression vectors.

Derivatives of L19:

L19: no further terminal modification

L19 His: C-terminal His ₆ domain (His tag) for Ni chelate chromatography and radioisotope binding

AP38: C-terminal GlyGlyGlyCys domain for binding (via Cys) of substances (eg, radioisotopes) that can be used for treatment and diagnosis

AP39: C-terminal GlyGlyGlyCysAla domain for binding (via Cys) of substances (eg, radioisotopes) that can be used for treatment and diagnosis

L19-GlyCysGlyCys: C-terminal GlyCysGlyCys domain for binding (via Cys) of substances (eg, radioisotopes) that can be used for treatment and diagnosis

L19-GlyCysGlyCysAla: C-terminal GlyCysGlyCysAla domain for binding (via Cys) of substances (eg, radioisotopes) that can be used for treatment and diagnosis

Recombinant Preparation of L19 Derivatives:

The L19 derivatives described were prepared in prokaryotic and eukaryotic expression systems.

a) this. L19 Preparation in E. Coli

DNA sequences encoding various L19 derivatives (AP38, AP39, L19-GlyCysGlyCys, L19-GlyCysGlyCysAla, L19, L19His) can be obtained from prokaryotic expression vectors (pDN5, Pini et al., 1997) with IPTG-inducible promoters and empicillin resistance labels. , J. Immunol.Methods 206: 171, Pini et al., 1998, J. Biol. Chem. 273: 21769; pET, Novagen). To enable secretion of the recombinant protein into the periplasm, this vector was used to prepare an expression cassette in which the Pel B signal sequence was fused to the N-terminus of the scFv. this. E. coli (TG1, BL21DE3 and HB2151) was transformed with this expression vector, and then a stable production strain was obtained by empicillin selection. To prepare the scFv, these strains were cultured in the presence of 1% glucose in the growth phase (37 ° C.) to inhibit the promoter. ScFv expression in culture was induced by IPTG addition and incubation at 30 ° C. for up to 16 hours. Soluble and antigen-binding scFv materials. It could be isolated from complete extracts of E. coli strains, from periplasmic fractions or from culture supernatants which proved particularly efficient in terms of purification and yield. Preparation was carried out in shake flasks and fermentors with culture volumes up to 10 liters.

b) pichia pastoris ( Pichia pastoris L19 derivatives in

For production in yeast Pichia pastoris L19His, AP38, AP39, L19-GlyCysGlyCys and L19-GlyCysGlyCysAla-encoding DNA sequences were amplified by PCR. E. coli and expression vectors were cloned into pPIC9K and pGAP (Invitrogen). For expression of heterologous genes, pPIC9K contains a methanol-inducible promoter (AOX1) and pGAP contains a homeostatic promoter of the GAPDH enzyme. In addition, these vectors each contain a signal sequence (in yeast α factor) for the expression and secretion of geneticin resistance genes and zeocin resistance genes and recombinant products for selection / amplification of foreign genes. The AP39 expression cassette used encodes a fusion protein (αfactor signal + L19 derivative) containing only Kex2 cleavage sites, but not other cleavage sites of native αfactor processing for signal sequence removal. Stable transfected PP clones were subjected to electroporation of the linearized vector to Pichia pastoris strains (eg pPIC9K-AP39 into strain GS115, pGAP-AP39 into strain X33) and subsequent agents. Obtained by netisin or zeocin screening. It was possible to use these clones to prepare the L19 derivatives as soluble secreted proteins. Clones were incubated at 30 ° C. in BMGY medium or basal mineral medium. Methanol was added to the pPIC based clones for promoter induction during the expression phase. The recombinant product had exactly processed terminal and high antigen-binding activity. Yields obtained (liter of unpurified bioproduct / culture supernatant) were as follows depending on culture conditions and method control: for example, pPIC9K-AP39 / GS115 (shaking flask 5 mg / l, fermenter 10-15 mg / l); pGAP-AP39 / X33 (shaking flask 30-40 mg / l, fermenter 100-250 mg / 1).

L19 derivatives were subjected to affinity chromatography (r Protein A, Streamline Pharmacia or ED B antigen column) and subsequent size exclusion chromatography to Pichia pastoris or E. coli . Purification from E. coli culture supernatant. The purified AP39 fraction used for further processing had homodimeric structure (covalently linked almost all of the subunits) and high antigen-binding activity.

Example 2a

Synthesis of Reduced AP38 [Reduced L19- (Gly) ₃ -Cys-OH]

50 μl TCEP-solution (14.34 mg TCEPxHCl / 5 ml aqueous Na ₂ HPO ₄ , 0.1 M, pH = 7.4) in 240 μg (4.29 nmol) SS-dimer AP38 solution in 156 μl PBS (phosphate buffered saline) / 10% glycerin Was added. The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product demonstrated quantitative conversion of SS-dimer AP38 to SH-monomer AP38.

Yield: 79.4 μg / 220 μl PBS (33.1%).

Example 2b

Tc-99m-AP38 [Tc-99m-L19- (Gly) ₃ -Cys-OH] Synthesis

2.37 mg disodium-L-tartrate was placed in a small bottle, then 79.4 μg reduced AP38 in 220 μl PBS was added and the solution diluted with 100 μl aqueous Na ₂ HPO ₄ -buffer (1M, pH = 10.5). 50 μl Tc-99m generator eluent (24h) and 10 μl SnCl ₂ -solution (5 mg SnCl ₂ / 1ml 0.1M HCl) were added. The reaction mixture was shaken at 37 ° C. for half hour. 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.

Immune Reactivity: 88.7%

Example 3a

Reduced AP39 [reduced L19- (Gly) ₃ -Cys-Ala-OH] Synthesis

To a solution of 240 μg (4.29 nmol) SS-dimer AP39 in 135 μl PBS / 10% glycerin was added 50 μl TCEP-solution (14.34 mg TCEP × HCl / 5 ml aqueous Na ₂ HPO ₄ , 0.1M, pH = 7.4). The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated product demonstrated quantitative conversion of SS-dimer AP39 to SH-monomer AP39.

Yield: 135.9 μg / 180 μl PBS (56.2%).

Example 3b

Tc-99m-AP39 [Tc-99m-L19- (Gly) ₃ -Cys-Ala-OH] Synthesis

4.2 mg disodium-L-tartrate was placed in a small bottle and 135.9 μg reduced AP39 in 180 μl PBS was added and the solution diluted with 100 μl aqueous Na ₂ HPO ₄ -buffer (1M, pH = 10.5). Tc-99m generator eluant 100㎕ (24h) and 10㎕ SnCl ₂ - A solution _{(5mg SnCl 2 / 1ml 0.1M HCl} ) were added thereto. The reaction mixture was shaken at 37 ° C. for half hour. 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).

Specificity: 21.4 MBq / nmol.

Immune Reactivity: 96.4%

Example 4

Re-188-AP38 [Re-188-L19- (Gly) ₃ -Cys-OH] Synthesis

2.37 mg disodium-L-tartrate was placed in a small bottle, then 112 μg reduced AP38 in 310 μl PBS was added and the solution diluted with 100 μl aqueous Na ₂ HPO ₄ -buffer (1M, pH = 10.5). 100㎕ Re-188 generator eluent and 50㎕ SnCl ₂ - A solution _{(5mg SnCl 2 / 1ml 0.1M HCl} ) were added. The reaction mixture was shaken at 37 ° C. for an hour and a half. Re-188-labeled AP38 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).

Radiochemical yield: 28.3%.

Radiochemical purity: 91.1% (SDS-PAGE).

Specificity: 15.3 MBq / nmol.

Immune Reactivity: 89.9%

Example 5

Re-188-AP39 [Re-188-L19- (Gly) ₃ -Cys-Ala-OH] Synthesis

2.37 mg disodium-L-tartrate was placed in a small bottle and then 112 μg reduced AP39 in 303 μl PBS was added and the solution diluted with 100 μl aqueous Na ₂ HPO ₄ -buffer (1M, pH = 10.5). 100㎕ Re-188 generator eluent and 50㎕ SnCl ₂ - A solution _{(5mg SnCl 2 / 1ml 0.1M HCl} ) were added. The reaction mixture was shaken at 37 ° C. for an hour and a half. Re-188-labeled AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).

Radiochemical yield: 33.5%.

Radiochemical purity: 92.3% (SDS-PAGE).

Specific activity: 18.5 MBq / nmol.

Immune Reactivity: 92.5%

Example 6a

Synthesis of Reduced L19-Gly-Cys-Gly-Cys-OH

75 μl TCEP-solution (14.34 mg TCEP × HCl / 5 ml aqueous Na ₂ HPO ₄ , 0.1 M in 240 μg (4.29 nmol) SS-dimer L19-Gly-Cys-Gly-Cys-OH solution in 160 μl PBS / 10% glycerin , pH = 7.4) was added. The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer L19-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 demonstrated quantitative conversion of SS-dimer L19-Gly-Cys-Gly-Cys-OH to SH-monomer L19-Gly-Cys-Gly-Cys-OH.

Yield: 80.4 μg / 210 μl PBS (33.5%).

Example 6b

Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-OH

2.37 mg disodium-L-tartrate was placed in a small bottle, followed by addition of 80.4 μg reduced L19-Gly-Cys-Gly-Cys-OH in 210 μl PBS and the solution was added to 100 μl aqueous Na ₂ HPO ₄ -buffer ( 1M, pH = 10.5). 50 μl Tc-99m generator eluent (24h) and 10 μl SnCl ₂ -solution (5 mg SnCl ₂ / 1ml 0.1M HCl) were added. The reaction mixture was shaken at 37 ° C. for half hour. Tc-99m-labeled L19-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.

Immune Reactivity: 89.7%

Example 7a

Synthesis of Reduced L19-Gly-Cys-Gly-Cys-Ala-OH

75 μl TCEP-solution (14.34 mg TCEP × HCl / 5 ml aqueous Na ₂ HPO ₄ , in 240 μg (4.29 nmol) SS-dimer L19-Gly-Cys-Gly-Cys-Ala-OH solution in 155 μl PBS / 10% glycerin 0.1M, pH = 7.4) was added. The reaction mixture was shaken slowly for 1 hour at room temperature. SH-monomer L19-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 demonstrated quantitative conversion of SS-dimer L19-Gly-Cys-Gly-Cys-Ala-OH to SH-monomer L19-Gly-Cys-Gly-Cys-Ala-OH.

Yield: 81.2 μg / 215 μl PBS (33.8%).

Example 7b

Synthesis of Tc-99m-L19-Gly-Cys-Gly-Cys-Ala-OH

2.37 mg disodium-L-tartrate was placed in a small bottle, followed by 81.2 μg reduced L19-Gly-Cys-Gly-Cys-Ala-OH in 215 μl PBS and the solution was added to 100 μl aqueous Na ₂ HPO ₄ −. Diluted with buffer (1M, pH = 10.5). 50 μl Tc-99m generator eluent (24h) and 10 μl SnCl ₂ -solution (5 mg SnCl ₂ / 1ml 0.1M HCl) were added. The reaction mixture was shaken at 37 ° C. for half hour. 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%.

Radiochemical purity: 93.5% (SDS-PAGE).

Specific activity: 19.1 MBq / nmol.

Immune Reactivity: 88.7%

Example 8a

Synthesis of Reduced AP39 for Specific Conjugation of EDTA, CDTA, TETA, DTPA, TTHA, HBED, DOTA, NOTA, and D03A Chelating Agents to Cysteine-SH Groups

50 μl TCEP-solution (14.34 mg TCEP × HCl / 5 ml aqueous Na ₂ HPO ₄ , 0.1M, pH = 7.4) was added to a 400 μg (7.1 nmol) AP39 solution in 450 μl PBS. The reaction mixture was shaken slowly at 37 ° C. for 1 hour. The 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 demonstrated complete conversion from AP39 to reduced AP39.

Yield: 140 μg / 200 μl (35%).

Example 8b

MX-DTPA-maleimide (1,4,7-triaza-2- (N-maleimido ethylene p -Amino) benzyl-1,7-bis (carboxymethyl) -4-carboxymethyl 6-methyl heptane)

512 mg (1 mmol) {[3- (4-amino-phenyl) -2- (bis-carboxymethyl-amino) -propyl]-[2- (bis-carboxymethyl-amino) -propyl] -amino} -acetic acid ( Macrocyclics Inc., Dallas, Texas, USA, and 707 mg (7 mmol) triethylamine were dissolved in 3 ml dry DMF. 400 mg (1.5 mmol) 3- (2,5-dioxo-2,5-dihydro-pyrrole-1-yl) -propionic acid 2,5-dioxo-pyrrolidin-1-yl ester in 1 ml dry DMF ( Aldrich). The solution was stirred at 50 ° C. for 5 hours. 30 ml diethyl ether was added slowly. The reaction mixture was further stirred for 30 minutes. The precipitate was collected by filtration. The crude product was purified by RP-HPLC (acetonitrile-: water-: trifluoroacetic 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 In-111-MX-DTPA-maleimide-S (Cys) -AP39-R

(R = reduced)

1,4,7-triaza-2- (N-maleimido ethylene p -amino) benzyl-1 dissolved in 50 μl of 140 μg (5 nmol) AP39-R in 200 μl sodium acetate buffer (0.1M, pH 5) The reaction was carried out at 37 ° C. for 3 hours with, 7-bis (carboxymethyl) -4-carboxymethyl 6-methylheptane (0.25 mg DTPA-maleimide in 500 μl sodium acetate buffer 0.1M pH 5). The reaction mixture was slide-a-lyser 10,000 MWCO (Pierce Inc., Rockford, Ill.) At 2 × 1 h with 200 ml sodium acetate buffer (0.1 M, pH 6). Dialysis

80 μl [In-111] InCl ₃ solution (HCl, 1N, 40MBq, Amersham Inc.) was added and the reaction mixture was heated at 37 ° C. for 30 minutes. In-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.

Immune Reactivity: 86%

Example 9

Synthesis of In-111-MX-DTPA-ε-HN (Lys) -AP39

Dilute 200 μg (3.6 nmol) unreduced AP39 in 111 μl PBS with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO (200 M sodium borate buffer (0.1M, pH 8.5)). Pearce Inc., Rockford, Illinois) was dialyzed at 2x1h. 50 μl 1,4,7-triaza-2- ( p -isothiocyanato) benzyl-1,7-bis (carboxymethyl) -4-carboxymethyl-6-methylheptane (MX-DTPA) solution (500 0.33 mg MX-DTPA dissolved in μL sodium borate buffer 0.1M pH 8.5) was added and the reaction mixture was heated at 37 ° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) using slide-a-riser 10,000 MWCO (Pierce Inc., Rockford, Ill.) At 2x1h and 1x17h (overnight), respectively.

80 μl [In-111] InCl ₃ solution (HCl, 1N, 40MBq, Amersham Inc.) was added and the reaction mixture was heated at 37 ° C. for 30 minutes. In-111 labeled MX-DTPA-ε-HN (Lys) -AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).

Radiochemical yield: 70%.

Radiochemical purity: 85% (SDS-PAGE).

Inactivity: 7.6 MBq / nmol.

Immune Reactivity: 74%

Example 10

Synthesis of In-111-DOTA-C-benzyl-p-NCS-ε-HN (Lys) -AP39

Dilute 200 μg (3.6 nmol) non-reduced AP39 in 114 μl PBS with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO (200 M sodium borate buffer (0.1M, pH 8.5)). Pearce Inc., Rockford, Illinois) was dialyzed at 2x1h. 50 μl 1,4,7,10-tetraaza-2- ( p -isothiocyanato) benzyl cyclododecane-1,4,7,10-tetraacetic acid (benzyl-p-SCN-DOTA, macrocyclics Inc., Dallas, Texas) solution (1.5 mg benzyl-p-SCN-DOTA dissolved in 5 ml sodium borate buffer 0.1M pH 8.5) was added to the solution and the reaction mixture was heated at 37 ° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) using slide-a-riser 10,000 MWCO (Pierce Inc., Rockford, Ill.) At 2x1h and 1x17h (overnight), respectively.

80 μl [In-111] InCl ₃ solution (HCl, 1N, 40MBq, Amersham Inc.) was added and the reaction mixture was heated at 37 ° C. for 30 minutes. In-111 labeled DOTA-C-benzyl-p-NCS-ε-HN (Lys) -AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS).

Radiochemical yield: 74%.

Radiochemical purity: 94% (SDS-PAGE).

Inactivity: 12.3 MBq / nmol.

Immune Reactivity: 73%

Example 11

Synthesis of Y-88-MX-DTPA-ε-HN (Lys) -AP39

Dilute 200 μg (3.6 nmol) unreduced AP39 in 115 μl PBS with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO (200 mL sodium borate buffer (0.1M, pH 8.5)). Pearce Inc., Rockford, Illinois) was dialyzed at 2x1h. 50 μl MX-DTPA solution (0.33 mg MX-DTPA dissolved in 500 μl sodium borate buffer 0.1M pH 8.5) was added and the reaction mixture was heated at 37 ° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) using slide-a-riser 10,000 MWCO (Pierce Inc., Rockford, Ill.) At 2x1h and 1x17h (overnight), respectively.

100 μl [Y-88] YCl ₃ solution (HCl, 1N, 75MBq, Oak Ridge National Lab.) Was added and the reaction mixture was heated at 37 ° C. for 30 minutes. Y-88 labeled MX-DTPA-ε-HN (Lys) -AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, eluent: PBS).

Radiochemical yield: 65%.

Radiochemical purity: 93% (SDS-PAGE).

Specificity: 10.2 MBq / nmol.

Immune Reactivity: 72%

Example 12

Synthesis of Lu-177-DOTA-C-benzyl-p-NCS-ε-HN (Lys) -AP3

Dilute 200 μg (3.6 nmol) unreduced AP39 in 110 μl PBS with 300 μl sodium borate buffer (0.1M, pH 8.5) and slide-a-riser 10,000 MWCO (200 mL sodium borate buffer (0.1M, pH 8.5)). Pearce Inc., Rockford, Illinois) was dialyzed at 2 x 1h. 50 μl benzyl-p-SCN-DOTA solution (1.5 mg dissolved in 5 ml sodium borate buffer 0.1M pH 8.5) was added and the reaction mixture was heated at 37 ° C. for 3 hours. The reaction mixture was dialyzed with 200 ml sodium acetate buffer (0.1M, pH 6.0) using slide-a-riser 10,000 MWCO (Pierce Inc., Rockford, Ill.) At 2x1h and 1x17h (overnight), respectively.

200 μl [Lu-177] LuCl ₃ solution (HCl, 1N, 80MBq, NRH-Petene (NRH-Petten, Netherlands)) was added and the reaction mixture was heated at 37 ° C. for 30 minutes. Lu-177 labeled DOTA-C-benzyl-p-NCS-ε-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.

Immune Reactivity: 71%

Example 13

Tracheal distribution and excretion of Tc-99m-AP39 expressed in Pichia pastoris after a single intravenous injection into tumor-bearing nude mice

The material of the invention was injected intravenously at a dose of about 74 kBq into F9 (teratoma) -bearing animals (about 25 g body weight). Radioactivity concentrations in various organs and radioactivity of manure were measured using a γ counter at various times after administration of the substance. In addition, the tumor to blood ratio was measured at various times based on the concentration of the substance of the invention in the tumor and blood.

Biodistribution (mean ± SD, n = 3) of Tc-99m-AP39 in F9 (morphologic cancer) -bearing nude mice is shown in Table 2:

Dose% / tissue g Dose% / tissue g Dose% / tissue g 1h p.i. 5h p.i. 24h p.i. Pancreas 1.97 ± 0.018 0.53 ± 0.07 0.31 ± 0.08 liver 1.91 ± 0.046 0.77 ± 0.07 0.26 ± 0.01 kidney 19.21 ± 0.70 4.35 ± 0.082 1.32 ± 0.10 lungs 3.43 ± 1.01 1.41 ± 0.032 0.96 ± 0.23 Stomach without contents 1.55 ± 0.043 1.35 ± 0.22 0.48 ± 0.10 A chapter with contents 1.42 ± 0.10 1.26 ± 0.34 0.29 ± 0.05 tumor 10.72 ± 3.21 5.13 ± 1.45 3.48 ± 1.28 blood 3.03 ± 0.32 0.57 ± 0.11 0.11 ± 0.01

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

Dose% 24h p.i. Yo 80.63 ± 3.33 credit 3.94 ± 0.17

Tumor-to-blood ratio (mean ± SD, n = 3) of Tc-99m-AP39 in F9 (malignant cancer) -bearing nude mice is shown in FIG. 2.

The results of the present invention show good discharge simultaneously with the good potential of the material of the present invention for accumulation in solid tumors.

Example 14

Organ Distribution of In-111-MX-DTPA-ε-HN (Lys) -AP39 After Single Intravenous Infusion into Tumor-bearing Nude Mice

The material of the invention was injected intravenously at a dose of about 48 kBq into F9 (teratoma) -bearing animals (about 25 g body weight). Radioactivity concentrations in various organs and radioactivity of manure were measured using a γ counter at various times after administration of the substance.

Biodistribution (mean ± SD, n = 3) of In-111-MX-DTPA-ε-HN (Lys) -AP39 in F9 (morphocarcinoma) -bearing nude mice is shown in Table 4:

Dose% / tissue g 1h p.i. 3h p.i. 24h p.i. Pancreas 1.94 ± 0.49 1.28 ± 0.13 1.18 ± 0.24 liver 2.61 ± 1.32 2.59 ± 0.36 2.26 ± 0.75 lungs 1.52 ± 1.57 2.36 ± 0.30 0.76 ± 0.21 Stomach without contents 1.44 ± 0.81 1.40 ± 0.31 0.65 ± 0.28 A chapter with contents 5.05 ± 5.26 1.07 ± 0.34 0.67 ± 0.11 tumor 12.90 ± 4.81 7.44 ± 1.34 4.33 ± 0.84 blood 5.55 ± 1.89 1.80 ± 0.20 0.11 ± 0.02

Tumor-to-blood ratio (mean ± SD, n = 3) of In-111-MX-DTPA-ε-HN (Lys) -AP39 in F9 (teratoma) -bearing nude mice is shown in Table 5.

1h p.i. 3h p.i. 24h p.i. Tumor to Blood Ratio 2.76 ± 2.00 4.16 ± 0.75 36.36 ± 3.78

The results of the present invention show an excellent biodistribution and tumor to blood ratio with the excellent potential of the material of the present invention for accumulation in solid tumors.

Example 15

Imaging of Tc-99m-AP39 Expressed in Pchia Pastoris after One Intravenous Injection into Tumor Nude Mice

The substance of the present invention was injected intravenously at a dose of about 9.25 MBq into an F9 (malignant cancer) -bearing animal (about 25 g body weight). Gamma-camera imaging was performed at various times after administration of the substance.

Scintillation of Tc-99m-AP39 in F9 (teratomatous cancer) -bearing nude mice is shown in FIGS. 3 and 4. FIG. 3 shows the scintogram after 5 hours of substance injection and FIG. 4 shows the scintogram after 24 hours of substance injection.

The results of the present invention show the excellent potential of the material of the present invention for imaging of solid tumors.

SEQUENCE LISTING <110> Schering AG <120> New methods for diagnosis and treatment of tumours <130> 27041P_WOAS <140> <141> <150> EP02 000 315.8 <151> 2002-01-03 <150> US60 / 358702 <151> 2002-02-25 <160> 13 <170> Patent In Ver. 2.1 <210> 1 <211> 238 <212> PRT <213> Artificial Sequence <220> <221> SITE (222) (1) .. (116) <223> VH <220> <221> SITE <222> (117) .. (130) <223> Linker <220> <221> SITE (222) (131) .. (238) <223> VL <220> <221> VARIANT (222) (1) .. (238) <223> deletion, insertion and / or substitution of up to       30 amino acids <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 1 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser         115 120 125 Thr Gly Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser     130 135 140 Pro Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser 145 150 155 160 Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg                 165 170 175 Leu Leu Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg             180 185 190 Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg         195 200 205 Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg     210 215 220 Ile Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 225 230 235 <210> 2 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <220> <221> VARIANT (222) (1) .. (3) <223> xaa each independently represent any naturally       occuring amino acid <400> 2 Xaa Xaa Xaa Cys   One <210> 3 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <220> <221> VARIANT (222) (1) .. (3) <223> Xaa each independently represent any naturally       occuring amino acid <220> <221> VARIANT <222> (5) <223> Xaa represents any naturally occuring amino acid <400> 3 Xaa Xaa Xaa Cys Xaa   1 5 <210> 4 <211> 1 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <220> <221> REPEAT <222> (1) <223> His = (His) n, wherein n stands for an integer from 4       to 6 <400> 4 His   One <210> 5 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 5 Gly Gly Gly Cys   One <210> 6 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 6 Gly Cys Gly Cys   One <210> 7 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 7 Gly Gly Gly Cys Ala   1 5 <210> 8 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 8 Gly Cys Gly Cys Ala   1 5 <210> 9 <211> 247 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser         115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly     130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu                 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser             180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu         195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro     210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Ala Ala Ala Leu 225 230 235 240 Glu His His His His His His                 245 <210> 10 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser         115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly     130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu                 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser             180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu         195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro     210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Cys 225 230 235 240                                                                   <210> 11 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 11 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser         115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly     130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu                 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser             180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu         195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro     210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Gly Gly Cys 225 230 235 240 Ala     <210> 12 <211> 240 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser         115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly     130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu                 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser             180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu         195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro     210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Cys Gly Cys 225 230 235 240                                                                   <210> 13 <211> 241 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: recombinant       antibody fragment <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly   1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Phe              20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val          35 40 45 Ser Ser Ile Ser Gly Ser Ser Gly Thr Thr Tyr Tyr Ala Asp Ser Val      50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr  65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys                  85 90 95 Ala Lys Pro Phe Pro Tyr Phe Asp Tyr Trp Gly Gln Gly Thr Leu Val             100 105 110 Thr Val Ser Ser Gly Asp Gly Ser Ser Gly Gly Ser Gly Gly Ala Ser         115 120 125 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly     130 135 140 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 145 150 155 160 Phe Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu                 165 170 175 Ile Tyr Tyr Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser             180 185 190 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu         195 200 205 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Thr Gly Arg Ile Pro     210 215 220 Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Gly Cys Gly Cys 225 230 235 240 Ala

Claims (21)

aa) an antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and / or LCDR3 shown in Table 1 or an HCDR3 region having the same function as a peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions, and / or substitutions of 0 to 5 amino acids of or less than 6 amino acids of the LCDR3 region; ab) the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or the peptide according to SEQ ID NO: 1 Less than 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 LCDR3 region Variants thereof which are the deletion, insertion and / or substitution of up to 6 amino acids of; or ac) a variant of SEQ ID NO: 1 that is a deletion, insertion, and / or substitution of up to 30 amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and ba) the amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys (SEQ ID NO: 2), wherein Xaa ₁ , Xaa ₂ , and Xaa ₃ each independently represent any naturally occurring amino acid; or bb) Amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys-Xaa ₄ (SEQ ID NO: 3), wherein Xaa ₁ , Xaa ₂ , Xaa ₃ , and Xaa ₄ each independently represent any naturally occurring amino acid or bc) amino acid sequence (His) _n (SEQ ID NO: 4), where n represents an integer from 4 to 6 Wherein the C-terminus of aa), ab) or ac) comprises a peptide linked to the N-terminus of one of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 via peptide bonds compound. According to claim 1, wherein the amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -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 ) Compound. The method according to claim 1, wherein the amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys-Xaa ₄ (SEQ ID NO: 3) is the sequence Gly-Gly-Gly-Cys-Ala (SEQ ID NO: 7) or Gly-Cys-Gly- A compound that is Cys-Ala (SEQ ID NO: 8). The compound of claim 1, wherein _{n in} the amino acid sequence (His) _n (SEQ ID NO: 4) is 6. 6. The compound of any one of claims 1-4 conjugated to a radioisotope. A radioisotope of technetium, for example ^{94 m} Tc, ^{99 m} Tc, rhenium, for example ¹⁸⁶ Re, ¹⁸⁸ Re, or other isotopes, for example ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m In, ¹¹¹ In, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁵³ Sm, ¹⁶⁶ Ho, A compound conjugated to a radioisotope selected from ¹⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As and ¹⁸ F. The compound of claim 6, wherein said radioisotope is ^99m Tc or ¹⁸⁸ Re. 8. A compound according to any one of claims 1 to 7, wherein said peptide is in reduced form. A pharmaceutical composition comprising a physiologically acceptable adjuvant, carrier and / or diluent together with a compound according to any one of claims 1 to 8 as an active agent. The composition of claim 9, wherein at least 70% of the mouse is excreted through the kidney within 24 hours. The composition of claim 9 or 10, wherein the tumor to blood ratio is at least 5: 1 after 5 hours of administration to the mice. The composition according to any one of claims 9 to 11, which is for diagnostic use. The composition according to any one of claims 9 to 11 for therapeutic use. aa) an antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining region HCDR3 and / or LCDR3 shown in Table 1 or an HCDR3 region having the same function as a peptide according to SEQ ID NO: 1 Variants thereof that are deletions, insertions, and / or substitutions of up to 5 amino acids of and up to 6 amino acids of the LCDR3 region; ab) the antigen-binding site for the extracellular domain B (ED-B) of fibronectin comprising the complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 shown in Table 1 or the peptide according to SEQ ID NO: 1 Less than 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 LCDR3 region Variants thereof which are the deletion, insertion and / or substitution of up to 6 amino acids of; or ac) a variant of SEQ ID NO: 1 that is a deletion, insertion, and / or substitution of up to 30 amino acids having the same function as the sequence according to SEQ ID NO: 1 (L19) or the peptide according to SEQ ID NO: 1, and ba) the amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys (SEQ ID NO: 2), wherein Xaa ₁ , Xaa ₂ , and Xaa ₃ each independently represent any naturally occurring amino acid; or bb) Amino acid sequence Xaa ₁ -Xaa ₂ -Xaa ₃ -Cys-Xaa ₄ (SEQ ID NO: 3), wherein Xaa ₁ , Xaa ₂ , Xaa ₃ , and Xaa ₄ each independently represent any naturally occurring amino acid or bc) amino acid sequence (His) _n (SEQ ID NO: 4), where n represents an integer from 4 to 6 Wherein the C-terminus of aa), ab), or ac) is linked to the N-terminus of one of the sequences of SEQ ID NO: 2, SEQ ID NO: 3, or SEQ ID NO: 4 via a peptide bond For combining elements. 15. The radioisotope of claim 14, wherein the radioisotope is a radioisotope of technetium, for example ^94m Tc, ^99m Tc, rhenium, for example ¹⁸⁶ Re, ¹⁸⁸ Re, or other isotope, for example ²⁰³ Pb, ⁶⁷ Ga. , ⁶⁸ Ga, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m In, ¹¹¹ In, ⁹⁷ Ru, ⁶² Cu, ⁶⁴ Cu, ⁶⁷ Cu, ⁶⁸ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb, ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As and ¹⁸ F. Use according to claim 15, wherein the radioisotope is ^99m Tc or ¹⁸⁸ Re. A method for preparing a peptide as defined by any one of claims 1 to 4, wherein the peptide has the property of being expressed in eukaryotic cells, in particular yeast cells. 18. The method of claim 17, wherein said eukaryotic cells are Pichia pastoris cells. The method of claim 17 or 18, wherein the peptide is expressed constantly. The method of claim 17, wherein the N-terminus of the peptide is fused directly to the Kex2-cleavage site of the α-signal sequence. A kit for the preparation of a radiopharmaceutical comprising a physiologically acceptable adjuvant with a peptide as defined in any one of claims 1 to 8.