ZA200406162B - Conjugates comprising an antibody specific for theED-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

New methods for diagnosis and treatment of tumours

Description Field of the invention

The present invention relates to new methods for diagnosis and treatment - of tumours, using novel peptides for binding radionuclides.

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 (1 998):

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 Biotechnol., 15, 542 — 546; Neri et al. (1997), Nature Biotechnol., 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 (Castellani 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 '*I 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 the 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 pastoris has already been described (Marty et al., Protein Expression and Purification 21, 156 - 164 (2001)).

Further, radiolabeling of scFv antibody fragments with **™T¢ through a C- terminal cysteinyl peptide was described by George et al., Proc. Natl.

Acad. Sci. 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 aa) 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; ab) 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

IN 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 : ILCDR2 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 ba) an amino acid sequence Xaa,-Xaa,-Xaa,;-Cys (Seq. Id. No. 2), wherein Xaa,, Xaa,, and Xaa, each independently represent any naturally occuring amino acid or bb) an amino acid sequence Xaa,-Xaa,-Xaa,-Cys-Xaa, (Seq. Id.

No. 3), wherein Xaa;,, Xaa, Xaas;, and Xaa, each independently represent any naturally occuring amino acid or bc) an amino acid sequence (His), (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 *™Tc, ®*"Tc

Rhenium, such as '®°Re, '®®Re, or other isotopes, such as 2°Pb, Ga, %Ga,

433¢, “ge, 473, Hominy, Mn, Ru, 82Cu, 4Cu, 57Cu, 88Cu, 8oy, 88y, oy, 121Sn, Tb, '53Sm, "®Ho, "Rh, 77Lu, 72As and '®F. . 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 aa) 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; ab) the sequence of the antigen-binding site for the extra domain

B(ED-B) of fibronectin comprising complementarity-

S..20 oo --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. ld. No. 1, . and . ba) an amino acid sequence Xaa,-Xaa,-Xaa;-Cys (Seq. Id. No. 2), wherein Xaa,, Xaa,, and Xaa, each independently represent any naturally occuring amino acid or bb) an amino acid sequence Xaa;-Xaa,-Xaaz-Cys-Xaa, (Seq. Id.

No. 3), wherein Xaa, Xaa,, Xaay, and Xaa, each independently represent any naturally occuring amino acid or bc) an amino acid sequence (His), (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,

S-20 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): (VH)

EVOLLESGGG LVQPGGSLRL SCAASGFTFS

SFSMSWVRQA PGKGLEWVSS ISGSSGTTYY

ADSVKGRFTI SRDNSKNTLY LQMNSLRAED

TAVYYCAKPF PYFDYWGQGT LVTVSS i (Linker)

GDGSSGGSGG ASTG

(VL)

EIVLTQSPGT LSLSPGERAT LSCRASQSVS

SSFLAWYQQK PGQAPRLLIY YASSRATGIP

DRFSGSGSGT DFTLTISRLE PEDFAVYYCQ

QTGRIPPTFG QGTKVEIK

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, 12,13, 14,15, 16, 17, 18, 19, 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).

Region CDR length (aa) | Sequence Maximum (preferred) variations in sequence positions

HCDR1 SESMS 3 (2,1)

HCDR2 17 SISGSSGT |8

TYYADSYV (7,6,5,4,3,2,1)

KG

HCDR3 PFPYFDY 5(4,3,2,1)

LCDR1 12 RASQSVS |6(5,4,3,2,1)

SSFLA

LCDR2 YASSRAT |4(321)

LCDR3 10 CQQTGRIP|6(5,4,3,21)

PT

TT CT "Table 1 To Co I

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 K,; that is in the . subnanomolar range (i.e. less than 10°), measured with a BlAcore (see

WQ099/58570, Example 2 and Table 2). ’

Preferred amino acid sequences Xaa;-Xaa,-Xaa,;-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;-Xaa,-Xaa;-Cys-Xaa, (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), (Seq. Id. No. 4), those compounds wherein n stands for the integer 6 are preferred.

Preferred radioisotopes of Technetium or Rhenium are the isotopes %™Tg, ~ 20 *9™Tg¢, '®®Re and '®®Re. Most preferred is the radioisotope Tc. Co

Detailed Description of the Invention

The single-chain antibody fragment L19 (Seq. Id. No. 1) was previously labeled with '®°l to investigate the biodistribution of this compound in tumour-bearing mice (Tarli et al., Blood, Vol. 94, No. 1 (1999), p. 192 - 198). 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 L19 may be substantially improved when it is conjugated to a peptide ba), bb) or bg) and labelled with radioisotopes of Technetium or Rhenium. The isotope mTc 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 5s '®Re and "®Re is especially preferred (Hsieh, B.T., et al., Nucl. Med. Biol., 1999, 26(8), 967-972; 973-976, Zamora, 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. 1)

L19His: 1 EVQLLESGGG LVQOPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

~~ B51 ~~ 'ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED =~ =~

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASE! 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 SGGSGGASE! VLTQSPGTLS

LSPGERATLS

151 CRASQSVSSS FLAWYQQKPG QAPRLLIYYA SSRATGIPDR

FSGSGSGTDF

201 TLTISRLEPE DFAVYYCQQT GRIPPTFGQG TKVEIKGGGC (Seq. Id. No. 10)

AP39: 1 EVQLLESGGG LVOQPGGSLRL SCAASGFTFS SFSMSWVRQA

PGKGLEWVSS

51 ISGSSGTTYY ADSVKGRFTI SRDNSKNTLY LOMNSLRAED

TAVYYCAKPF

101 PYFDYWGQGT LVTVSSGDGS SGGSGGASE! VLTQSPGTLS “20° : ~“LSPGERATLS Co ST oo 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 oo | oo I Ca . (Seq. Id. No. 13)

The production of the peptides is described in detail in the following examples (see Experimental”).

The antibody fragment L19 was originally produced by expression in E. coli (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. pPIC9K). 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 metastases. 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 = radioisotopes’ as described above, e.g. radioisotopes of Technetium and

Rhenium, preferably the radionuclides %™Tc, %¥™Tc, '®®Re, and '88Re. 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 ®*™T¢ generator eluate or '®8Re 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 triamine pentaacetic acid (DTPA), cyclohexyl 1,2-diamine tetraaceticacid (CDTA), ethyleneglycol-O,0’-bis(2-aminoethyl)-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), 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-NH,-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. **Tc-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 2%], 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. **™Tc- labeled AP39 is 5:1 or more, preferably 8:1 or more, e.g. about 10 : 1 © 20 after 5 hours, whereas for L19 labeled with '?°|, this ratio is only about 3 : 1. This is an unexpected behaviour also compared to other scFv antibodies labeled with ®°™Tc which often show less favourable biodistribution characteristics. For example, Verhaar et al., J. Nuc. Med.,

Vol. 37(5), pp. 868 — 872, 1996, report a **"Tc-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 ®mTc-labeled AP39 (see example 13 below).

Further, the in vivo stability of the labeled peptides of the invention, e.g. mTs-labeled AP39 is much higher compared to the in vivo stability of L19 labeled with '#°l. The present inventors found that 2 hours after injection of a peptide, e.g. **"Tc-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 '*°I, 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. ®*"Tc-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. %mTc-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 L19, 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 In-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 In-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 100 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.

Experimental

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 et al., 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 (K;: 5.4 x 10" 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.

S20 L19 derivatives:

L19: without additional terminal modifications

L19 His: C-terminal His, domain (His tag), for Ni chelate chromatography 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 L19 derivatives

The L19 derivatives described were produced in prokaryotic and eukaryotic expression systems. a) L19 production in E. coli

The DNA 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, ~~" Novagen) 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% ) 25 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 16 h. 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 L19 derivatives in Pichia pastoris

L19His, AP38, AP39, L19-GlyCysGlyCys and L19-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, pPICIK 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

SE 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- 15 mg/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);-Cys-OH]

To a solution of 240 ug (4.29 nmol) S-S-dimeric AP38 in 156 ul PBS (phosphate buffered saline)/10% glycerine were added 50 ul TCEP-solution } (14.34 mg TCEP x HCI/5 ml aqueous Na,HPO,, 0.1 M, pH = 7.4). The reaction mixture was gently shaken for 1 h at room temperature. SH- monomeric 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- Co monomeric AP38.

Yield: 79.4 ug/220 ul PBS (33.1%).

Example 2b

Synthesis of Tc-99m-AP38 [Tc-99m-L19-(Gly),-Cys-OH] 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 79.4 ug reduced AP38 in 220 ul PBS and the solution was diluted with 100 pl aqueous Na,HPO,-buffer (1 M, pH = 10.5). 50 ul Tc-99m generator eluate (24 h) and 10 ul SnCl,-solution {5 mg SnCl,/1T 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 MBg/nmol.

Immunoreactivity: 88.7%

Example 3a

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

To a solution of 240 yg (4.29 nmol) S-S-dimeric AP39 in 135 ul PBS/10% glycerine were added 50 ul TCEP-solution (14.34 mg TCEP x HCIl/5 ml aqueous Na,HPO,, 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 ug/180 ul PBS (56.2%).

Example 3b

Synthesis of Tc-99m-AP39 [Tc-99m-L19-(Gly),;-Cys-Ala-OH] 4.2 mg disodium-L-tartrate were placed in a vial followed by addition of 135.9 ug reduced AP39 in 180 ul PBS and the solution was diluted with 100 ul aqueous Na,HPO,-buffer (1 M, pH = 10.5). 100 ul Tc-99m generator eluate (24 h) and 10 ul SnCl,-solution (5 mg SnCl,/1 ml 0.1 M

HCI) were added. The reaction mixture was shaken for 0.5 h at 37°C. Te- 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 MBg/nmol.

Immunoreactivity: 96.4% -- Example 4 - DEERE - So --

Synthesis of Re-188-AP38 [Re-188-L19-(Gly),-Cys-OH] 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 112 pg reduced AP38 in 310 ul PBS and the solution was diluted with 100 ub aqueous Na,HPO,-buffer {1 M, pH = 10.5). 100 ul Re-188 generator eluate and 50 ul SnCl,-solution (5 mg SnCl,/1 ml 0.1 M HCI) were added. x 25 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: 91.1% (SDS-PAGE). } Specific activity: 15.3 MBg/nmol.

Immunoreactivity: 89.9%

Example 5

Synthesis of Re-188-AP39 [Re-188-L19-(Gly),-Cys-Ala-OH] 2.37 mg disodium-L-tartrate were placed in a vial followed by addition of 112 ug reduced AP39 in 303 ul PBS and the solution was diluted with 100 ul aqueous Na,HPO,-buffer (1 M, pH = 10.5). 100 ul Re-188 generator eluate and 50 ul SnCl,-solution (5 mg SnCl,/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: 33.5%. . Radiochemical purity: 92.3% (SDS-PAGE). -.

Specific activity: 18.5 MBqg/nmol.

Immunoreactivity: 92.5%

Example 6a

Synthesis of reduced L19-Gly-Cys-Gly-Cys-OH

To a solution of 240 ug (4.29 nmol) S-S-dimeric L19-Gly-Cys-Gly-Cys-OH in 160 ul PBS/10% glycerine were added 75 ul TCEP-solution (14.34 mg

TCEP x HCI/5 ml aqueous Na,HPO,, 0.1 M, pH = 7.4). The reaction mixture was gently shaken for 1 h at room temperature. SH-monomeric

L19-Gly-Cys-Gly-Cys-OH was purified by gel-chromatography using a NAP- column (Amersham, Eluent: PBS). SDS-PAGE analysis of the isolated } product proofed the quantitative transformation of S-S-dimeric L19-Gly- 5 Cys-Gly-Cys-OH to SH-monomeric L19-Gly-Cys-Gly-Cys-OH.

Yield: 80.4 ug/210 ul 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 ug reduced L19-Gly-Cys-Gly-Cys-OH in 210 ul PBS and the solution was diluted with 100 ul aqueous Na,HPO,-buffer (1 M, pH = 10.5). 50 Ml

Tc-99m generator eluate (24 h) and 10 ul SnCl,-solution (5 mg SnClL,/1 mi 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-OH was purified by gel-

SE chromatography using a'NAP-5 column (Amersham, Eluent: PBS). C7

Radiochemical yield: 37.7%.

Radiochemical purity: 91.5% (SDS-PAGE).

Specific activity: 19.7 MBg/nmol.

Immunoreactivity: 89.7%

Example 7a

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

To a solution of 240 ug (4.29 nmol) S-S-dimeric L1 9-Gly-Cys-Gly-Cys-Ala-

OH in 155 ul PBS/10% glycerine were added 75 ul TCEP-solution (14.34 mg TCEP x HCI/5 ml aqueous Na,HPO,, 0.1 M, pH = 7.4). The reaction mixture was gently shaked for 1 h at room temperature. SH-monomeric

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 proofed the quantitative transformation of S-S-dimeric

L19-Gly-Cys-Gly-Cys-Ala-OH to SH-monomeric L1 9-Gly-Cys-Gly-Cys-Ala-

OH.

Yield: 81.2 yg/215 ul 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 ug reduced L19-Gly-Cys-Gly-Cys-Ala-OH in 215 ul PBS and the solution was diluted with 100 ul aqueous Na,HPO,-buffer (1 M, pH = 10.5). 50 p41 Tc-99m generator eluate (24 h) and 10 ul SnCl,-solution (5 mg

SnCl,/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%.

Radiochemical purity: 93.5% (SDS-PAGE).

Specific activity: 19.1 MBg/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 50ul TCEP-solution (14.34mg TCEPxHCI/5m! aqueous Na,HPO,, 0.1M, pH = 7.4) were added to a solution of 400xg (7.1 nmol) AP39 in 450ul 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: 140ug/200ul (35%).

Example 8b

Synthesis of 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) of {[3-(4-Amino-phenyl)-2-(bis-carboxymethyl-amino)- ] 25 propyll-[2-(bis-carboxymethyl-amino)-propyll-amino}-acetic acid (Macrocyclics Inc. Dallas, TX, U.S.A.) and 707 mg (7 mmol) triethylamine were dissolved in 3 ml dry DMF. 400 mg (1,5 mmol) of 3-(2,5-Dioxo-2,5- dihydro-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 In-111-MX-DTPA-Maleimide-S(Cys)-AP39-R (R = reduced) 140 ug (5 nmol) AP39-R in 200 ul of sodium acetate buffer (0.1M, pH 5) were reacted with 50ul of dissolved 1,4,7-triaza-2-(N-maleimido ethylene p-amino)benzyl-1,7-bis(carboxymethyl)-4-carboxymethyl6-methylheptane (0,25mg DTPA-Maleimide in 500ul sodium acetate buffer 0.1M pH 5) for 3 h at 37°C. The reaction mixture was dialyzed 2 x 1 h with 200ml! of © sodium acetate buffer (0.1M, pH 6) employing a Slide-A-Lyzer 10,000 ~~

MWCO (Pierce Inc., Rockford, IL, U.S.A.). 80 ul [In-111]1InCl; solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37°C for 30 min. 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 MBg/nmol.

Immunoreactivity: 86 %

Example 9

Synthesis of in-1 11-MX-DTPA-e-HN(Lys)-AP39 200 ug (3.6 nmol) non-reduced AP39 in 111 ul PBS were diluted with 300

MI 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 ul of 1,4,7-triaza-2-(p- isothiocyanato)benzyl-1 .7-bis(carboxymethyl)-4-carboxymethyl-6-methyl heptane (MX-DTPA) solution (0.33 mg MX-DTPA dissolved in 500 Hl 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 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 ul [in-111]InCl; solution (HCI, 1N, 40 MBq, Amersham Inc.) were added - and the reaction mixture was heated -at 37°C for 30 min. In=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 MBqg/nmol.

Immunoreactivity: 74 %

Example 10

Synthesis of In-111 -DOTA-C-Benzyl-p-NCS -e-HN(Lys)-AP39 200 ug (3.6 nmol) non-reduced AP39 in 114 ul PBS were diluted with 300

Hl 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 ul of 1,4,7,10-tetraaza-2-(p- 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 2x 1 hand 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 ul [In-111]inCl; solution (HCI, 1N, 40 MBq, Amersham Inc.) were added and the reaction mixture was heated at 37°C for 30 min. In-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 MBg/nmol.

Immunoreactivity: 73 %

Example 11

Synthesis of Y-88-MX-DTPA-e-HN(Lys)-AP39 200 ug (3.6 nmol) non-reduced AP39 in 115 ui PBS were diluted with 300

Hl of sodium borate buffer (0.1M, pH 8.5) and dialyzed 2 x 1 h with 200m! 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 ul of MX-DTPA solution (0.33 mg MX-DTPA dissolved in 500 ul 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 hand 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.). 100 ul [Y-88]YCI, 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-e-HN(Lys)-AP39 was purified by gel-chromatography using a NAP-5 column (Amersham, Eluent: PBS). A

Radiochemical yield: 65 %.

Radiochemical purity: 93 % (SDS-PAGE).

Specific activity: 10.2 MBg/nmol.

Immunoreactivity: 72 %

Example 12

Synthesis of Lu-177 -DOTA-C-Benzyl-p-NCS-e-HN(Lys)-AP3 200 ug (3.6 nmol) non-reduced AP39 in 110 ul PBS were diluted with 300

Hu! 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 ul 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 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.). 200 ul [Lu-1771LuCI3 solution (HCI, TN, 80 MBq, 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-¢-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 MBg/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 (bodyweight about 25 gl.

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:

CL % ofdose/g | % ofdose/g | % ofdose/g - of tissue of tissue of tissue 1.97 +0.018 | 0.563 +0.07 0.31 +£0.08 1.91 =+0.046 | 0.77 +£0.07 0.26 +£0.01 19.21 £0.70 4.35 +0.082 1.32 +£0.10 3.43 =*=1.01 1.41 +0.032 0.96 +£0.23 . Stomach 1.56 +0.043 | 1.35 +£0.22 0.48 £0.10 without contents

Intestine with 1.42 +£0.10 1.26 +£0.34 0.29 +£0.05 N contents . 10.72 +£3.21 5.13 +£1.45 3.48 +£1.28 3.03 %0.32 (057 +0.11 |0.11 £0.01

Table 2

The excretion of Tc-99m-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean = SD, n=3) is shown in Table 3: T ofdoss 1 @mpa 80.63 £3.33 94 20.17

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 In-111-MX-DTPA-e-HN(Lys)-AP39 after a single i.v. injection into tumour-bearing nude mice

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 In-111-MX-DTPA-e-HN(Lys)-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean + SD, n=3) is shown in

Table 4: % dose / g of tissue 1h p.i. 3h p.i. 24h p.i. : Spleen’ 11.94 + 0.49 1.28 £ 0.13 [1.18 + 0.24" - 2.617 + 1.32 2.59 + 0.36 2.26 + 0.75 1.52 = 1.67 | 2.36 = 0.30 [0.76 = 0.21

Stomach 1.44 + 0.81 1.40 + 0.31 0.65 + 0.28 without contents

Intestine with 5.05 + 5.26 1.07 = 0.34 0.67 = 0.11 : 25 contents 12.90 = 4.81 7.44 + 1.34 4.33 + 0.84 : 5.56 + 1.89 1.80 + 0.20 0.11 £ 0.02

Table 4

. The tumour to blood ratio of In-111-MX-DTPA-e-HN(Lys)-AP39 in F9 (teratocarcinoma)-bearing nude mice (mean * SD, n=3) is shown in ’ Table 5.

Tumour to 2.76 + 2.00 4.16 + 0.75 36.36 + 3.78 blood ratio

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 g). Gamma-camera imaging is carried out at various times after administration of the substance. 25 ’ 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 i substance of the invention for imaging solid tumours.

Claims (33)

Claims

1. A compound comprising a peptide comprising : aa) an antigen-binding site for the extra domain B (ED-B) of fibronectin comprising complementarity-determining regions HCDRS3 and/or LCDR3 as shown in Table 1 or a variation thereof that is a deletion, insertion and/or O substitution of up to 5 amino acids for the HCDR3 region and up to 6 amino acids for the LCDRS3 region which has the same function as a peptide according to Seq. Id. No. 1; ab) an antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity-determining regions HCDR1, HCDR2, HCDRS3, 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; or 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, an which has the same function as a peptide according to Seq. Id. No. 1, and ba) an amino acid sequence Xaa,-Xaa,-Xaa;-Cys (Seq. Id. No. 2), wherein Xaa,, Xaa, and Xaa, each independently represent any naturally occuring amino acid or bb) an amino acid sequence Xaa,-Xaa,-Xaa;-Cys-Xaa, (Seq. ld. No. . 3), wherein Xaa,, Xaa,, Xaa,;, and Xaa, each independently represent any naturally occuring amino acid or } bc) an amino acid sequence (His), (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. ld. No. 2, Seq. Id. No. 3 or Seq. id. No. 4 via a peptide bond.

2. The compound according to claim 1, wherein the amino acid sequence Xaa,-Xaa,-Xaa;-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 according to claim 1, wherein the amino acid sequence Xaa,-Xaa,-Xaay-Cys-Xaa, (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 according to claim 1, wherein n in the amino acid sequence (His), (Seq. Id. No. 4) is 6.

5. The compound according to any one of claims 1-4 which is conjugated to a radioisotope. ) 25

6. The compound according to claim 6 which is conjugated to a radioisotope selected from a radioisotope of Technetium, such as %mTg, "Tc, Rhenium, such as '®Re, '%®Re, or other isotopes, such as 2°°Pb, ®’Ga, %%Ga, **Sc, **Sc, *’Sc, "In,

"MR 97Ru 82Cy 64Cu 7Cu 88Cu 86y 88y soy ST 181Th ] 183g m 166140 105RpH 77 u 2p and 18g

7. A compound according to claim 6, wherein the radioisotope is 9¥mTc or '88Re.

8. The compound according to any one of claims 1-7, wherein the peptide is in reduced form.

9. A pharmaceutical composition comprising as an active agent a compound according to any one of claims 1-8 together with physiologically acceptable adjuvants, carriers and/or diluents.

10. The composition of claim 9 which is excreted to 70% or more via the kidneys within 24 hours in mice.

11. The composition of claim 9 or 10 having a tumour to blood oo ratio of 5:1 or more 5 h after administration in mice. .

12. The composition of any one of claims 9-11 for diagnostic applications.

13. The composition of any one of claims 9-11 for therapeutic ; applications.

14. Use of a peptide comprising . aa) an 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 amino acids for the HCDR3 region and up to 6 amino acids ; for the LCDR3 region which has the same function as a peptide 5 according to Seq. Id. No. 1; ab) an antigen-binding site for the extra domain B(ED-B) of fibronectin comprising complementarity-determining regions HCDR1, HCDR2, HCDR3, LCDR1, LCDR2Z 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 LCDRS3 region; which has the same function as a peptide according to Seq. ld. No. 1; or ac) a sequence according to Seq. !d. No. 1 (L19) or a variation of

Seq. id. No. 1 that is a deletion, insertion and/or substitution of } oo up to 30 amino acids, an which has the same function as a peptide according to Seq. ld. No. 1, oo and ba) an amino acid sequence Xaa,-Xaa,-Xaa;-Cys (Seq. Id. No. 2), wherein Xaa,, Xaa, and Xaa; each independently represent any naturally occuring amino acid or bb) an amino acid sequence Xaa,-Xaa,-Xaa;-Cys-Xaa, (Seq. Id. No. . 3), wherein Xaa,, Xaa,, Xaa;, and Xaa, each independently represent any naturally occuring amino acid or ) bc) an amino acid sequence (His), (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. ld. No. 2, Seq. Id. No. 3 or Seq. |d. No. 4 via a peptide bond, ) for binding a radioisotope.

15. The use according to claim 14, wherein the radioisotope is selected from a radioisotope of Technetium, such as S4mTe, 9mTe Rhenium, such as '%°Re, '® Re, or other isotopes, such as 203py, 87Ga, 68Ga, Ag, ge, 47ge, Tomy, in, Ru, 62Cu, 84Cu, Cu, 88Cu, by, 88y oy, 1218p, 181 Th, 153g, 1680, 105g, 77) u, "?As and "°F.

16. The use according to claim 15, wherein the radioisotope is 9mTe or '8%Re.

17. A process for the production of a peptide as defined in any one of claims 1-4, characterized in that the peptide is expressed in eukaryotic cells, particularly in yeast cells.

18. The process according to claim 17, wherein the eukaryotic cells are Pichia pastoris cells.

19. The process according to claim 17 or 18, wherein the peptide is expressed constitutively.

.

20. The process according to any one of claims 17-19, wherein the N-terminus of the peptide is directly fused to the Kex2- cleavage site from the a-signal sequence.

41 PCT/EQ03/00009

21. A kit for the production of radiopharmaceuticals comprising a peptide as defined in any one of claims 1-8, optionally together with physiologically acceptable adjuvants.

22. Use of a compound according to any one of claims 1-8, in the manufacture of a preparation for diagnosing or treating tumours.

23. Use of a compound according to any one of claims 1-8, in the manufacture of a preparation for use with a radioisotope in diagnosing or treating tumours.

24. A substance or composition for us in a method of diagnosing or treating tumours, said substance or composition comprising a compound according to any one of claims 1-8, and said method comprising administering said substance or composition.

25. A substance or composition for use with a radioisotope in a method of diagnosing or treating tumours, said substance of composition comprising a compound according to any one of claims 1-8, and said method comprising administering said substance or composition.

26. A compound according to any one of claims 1 to 8, substantially as herein described and illustrated.

27. A composition according to any one of claims 9 to 13, substantially as herein described and illustrated.

28. Use according to any one of claims 14 to 16, substantially as herein described and illustrated. AMENDED SHEET

42 PCT/EO03/00009

29. A process according to any one of claims 17 to 20, substantially as herein described and illustrated.

30. A kit according to claim 21, substantially as herein described and illustrated.

31. Use according to claim 22 or claim 23, substantially as herein described and illustrated.

32. A substance or composition for use in a method of diagnosis or treatment according to claim 24 or claim 25, substantially as herein described and illustrated.

33. A new compound, a new composition, a new use of a compound according to any one of claims 1 to 8 or as defined in claim 14, a new use of a compound according to any one of claims 1 to 8 and a radioisotope, or a substance or composition for a new use in a method of diagnosis or treatment, substantially as herein described. AMENDED SHEET