WO2011110490A1 - Process for the production of radioactively labelled scfv antibody fragments, kits and compositions - Google Patents

Abstract

The process for the production of radioactively labelled scFv-labelled antibody fragments is described, as well as compositions and kits for use of said fragments for diagnostic and treatment of several diseases.

Description

Process for the production of radioactively labelled scFv antibody fragments,

Kits and Compositions

The present invention refers to the production of radioactively scFv-labelled antibody fragments and compositions for use of said fragments for diagnosis and treatment of several diseases.

The present invention especially refers to the production of radioactively Tc- and/or Re-labelled scFv-labelled antibody fragments and compositions for use of said fragments for diagnosis and treatment of several diseases.

Recombinant antibody fragments and engineered variants are emerging as useful alternatives to monoclonal antibodies for diagnostic and therapeutic purposes

(Hollinger, P. and Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains. Nature Biotechnol., 23, 1 126-35). Single chain antibody fragments (scFv's) (Raag, R. and Whitlow, M. (1995) Single-chain Fvs. FASEB J., 9, 73-80), are good molecules for tumor imaging and radiotherapy, owing to their rapid blood clearance and superior tumor penetration relative to full antibodies. Technetium-99m (^99mTc ) has been proposed as an appropriate imaging isotope for scFv's (Verhaar, M.J., Keep, P.A., Hawkins, R.E., Robson, L, Casey, J.L., Pedley, B., Boden, J.A., Begent, R.H.J., and Chester, K.A. (1996) Technetium-99m radiolabeling using phage-derived single-chain Fv with C-terminal cysteine. J. Nucl. Med., 37, 868-72), due to its moderate half life (~6 hours), which is well-suited for the biological uptake rate and clearance of these antibody fragments. Several efforts to radiolabel scFv's with ^99mTc have been reported (Waibel R., Alberto R., Willuda J., Finnern R., Schibli R., Stichelberqer A., Eqli A., Abram U., Mach J.P., Pluckthun A., and Schubiqer P.A. (1999) Stable one-step technetium-99m labeling of His-tagged recombinant proteins with a novel Tc(l)-carbonyl complex. Nature Biotechnol., 17, 897-901 ; Goel A., Baranowska-Kortylewicz J., Hinrichs S.H., Wisecarver J., Paylinkova G., Augustine S.. Colcher P., Booth B.J.. and Batra S.K. (2001 ) ^99mTc-labeled divalent and tetravalent CC49 single-chain Fv's: novel imaging agents for rapid in vivo localization of human colon carcinoma. J. Nucl. Med., 42, 1519-27; Pietersz G.A., Patrick M.R., and Chester K.A. (1998) Preclinical characterization and in vivo imaging studies of an engineered recombinant technetium-99m-labeled metallothionein-containing anti- carcinoembryonic antigen single-chain antibody. J. Nucl. Med., 39, 47-56). In addition, isotopes of rhenium (e.g. ¹⁸⁸Re; half life = 17 hours or ¹⁸⁶Re; half life = 3.8 days), which is a chemical congener of technetium could be utilized with scFvs for radiotherapeutic purposes.

The success of a strategy to utilize Tc/Re for imaging or radiotherapy using scFvs will depend largely on the quality of the radiolabeling method. Direct methods are known in the prior art for adding radioactive Tc/Re to biological molecules (US 5,061 ,641 ), and have been applied to scFvs (Pietersz G.A., Patrick M.R., and Chester K.A.

(1998) Preclinical characterization and in vivo imaging studies of an engineered recombinant technetium-99m-labeled metallothionein-containing anti- carcinoembryonic antigen single-chain antibody. J. Nucl. Med., 39, 47-56). However direct methods often show instability of the radioactive compound in vitro (i.e. poor shelf life) and in vivo (i.e. release of free Tc / Re as pertechnetate / perrhenate in the patient). They have the additional disadvantage that the radiolabeling is not controlled in regard to site of attachment of the radioisotope, and in fact, the radioisotope can often add in more than one location on the molecule. The biological efficacy of the molecule can be affected if the radioisiotope adds to a portion of the targeting protein critical for binding.

The use of chelating agents (chelators) for radiolabeling targeting proteins with radiometals can overcome many of the disadvantages of direct radiolabeling methods. The chelators form stable metal complexes that attach the radiometal more reliably and stably onto the targeting molecule. In addition, the metal chelator can be added to an antibody or antibody fragment in a defined manner with several options for point of attachment. Chelators that are comprised of naturally-occurring amino acids (peptide chelators) are particularly advantageous for labeling proteins, because they can be incorporated into the protein during the recombinant production process.

A well-established peptide chelator that is useful for complexing Tc/Re is X-X-Cys (X = any natural amino acid preferably except proline). The X-X-Cys sequence utilizes 3 amide nitrogens (from the peptide bonds) and a thiol sulfur (from the cysteine thiol) to form an "N₃S" chelator capable of complexing Tc(V) or Re(V) oxo cores. Peptide chelators of the triamide-thiol type are well-established, as exemplified by the ^99mTc metal center of [^99mTcO]apcitide (AcuTect™), which is an approved

radiopharmaceutical for thrombus imaging (Francesconi, L.C., Zheng, Y., Bartis, J., Blumenstein, M., Costello, C, and De Rosch, M.A. Preparation and Characterization of [⁹⁹TcO] Apcitide: A Technetium Labeled Peptide. (2004) Inorg. Chem., 43, 2867- 2875). Methods for radiolabeling scFvs with ^99mTc using X-X-Cys chelators are described in the prior art. Verhaar et. al. incorporated Ala-Ala-Cys-His-His-His-His-His at the C-terminus of an anti-CEA scFv, and attempted radiolabeling with ^99mTc at the Ala-Ala-Cys chelator (Verhaar, M.J., Keep, P.A., Hawkins, R.E., Robson, L, Casey, J.L., Pedley, B., Boden, J.A., Begent, R.H.J., and Chester, K.A. (1996) Technetium-99m radiolabeling using phage-derived single-chain Fv with C-terminal cysteine. J. Nucl. Med., 37, 868-72). The radiolabeling of unreduced scFv by transchelation from ^99mTc glucarate gave only a low radiochemical yield (5-10%).

George et. al. incorporated Gly-Gly-Gly-Gly-Cys at the C-terminus of several scFvs, and achieved high radiolabeling yields (>97%) with ^99mTc at the Gly-Gly-Cys chelator (George, A.J.T., Jamar, F., Tai, M-S., Heelan, B.T., Adams, G.P., McCartney, J.E., Houston, L.L., Weiner L.M., Oppermann, H., Peters, A.M., and Huston, J.S. (1995) Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide. Proc. Natl. Acad. Sci., 92, 8358-62). In these studies, radiolabeling was effected at pH >9.5, by reducing ^99mTc

pertechnetate with stannous tin (Tin(ll) chloride) in the presence of a small amount of protein. Mild reduction of the scFv prior to radiolabeling presumably freed up the cysteine thiol for better complexation to the metal, leading to improved radiolabeling. Berndorff et. al. described a scFv targeting ED-B fibronectin that radiolabeled with ^99mTc through a C-terminal Gly-Gly-Cys(-Ala) chelator (Berndorff, D., Borkowski, S., Moosmayer, D., Viti, F., Mueller-Tiemann, B., Sieger, S., Friebe, M., Hilger C.S., Zardi, L, Neri, D., and Dinkelborg, L.M. (2006) Imaging of tumor angiogenesis using ^99mTc-labeled human recombinant anti-ED-B fibronectin antibody fragments. J. Nucl. Med., 47, 1707-1716). This scFv also utilized reduced protein and radiolabeled in high yield (>97%) through a high-pH (10.5) procedure with stannous tin.

The scFv format is comprised of V_H and V_L domains linked by a peptide sequence, and when that sequence is relatively short, it is expected to associate into a multimeric form, for example dimeric form, with increased avidity relative to the monomer (Holliger, P., and Hudson, P.J. (2005) Engineered antibody fragments and the rise of single domains. Nature Biotech., 23, 1 126-35. b) Kort, A.A., Dolezal, O., Power, B.E., and Hudson, P.J. (2001 ) Dimeric and trimeric antibodies: high avidity scFvs for cancer targeting. Biomol. Eng., 18, 95-108). Furthermore, an equilibrium mixture of the different forms can be realized (Arndt, K.M., Mueller, K.M., and

Plueckthun, A. (1998) Factors influencing the dimer to monomer transition of an antibody single-chain Fv fragment. Biochemistry, 37, 12918-26). For tumor-targeting, increased avidity along with its longer blood residence time favors the multimer species over the monomer. In studies comparing radiolabeled dimeric vs.

monomeric forms of the same scFv, the superior biological properties of the non- covalent dimeric species relative to the monomeric species have been confirmed (Nielsen, U.B., Adams, G.P., Weiner, L.M., and Marks, J.D. (2000) Targeting of bivalent anti-ErbB2 diabody antibody fragments to tumor cells is independent of the intrinsic antibody affinity. Cancer Res., 60, 6434-40; Wu A.M.. Chen W.. Raubitschek A., Williams L.E.. Neumaier M.. Fischer R.. Hu S.Z.. Odom-Marvon T., Wong J.Y.. and Shively J.E. (1996) Tumor localization of anti-CEA single-chain Fvs: improved targeting by non-covalent dimers. Immunotechnol., 2, 21 -36; Huang, B-C, Foote, L.J., Lankford, T.K., Davern, S.M., McKeown, C.K., and Kennel, S.J. (2005) A diabody that dissociates to monomer forms at low concentration: effects on binding and tumor targeting. Biochem. Biophys. Res. Comm., 327, 999-1005).

In the studies described above (George, A.J.T., Jamar, F., Tai, M-S., Heelan, B.T., Adams, G.P., McCartney, J.E., Houston, L.L., Weiner L.M., Oppermann, H., Peters, A.M., and Huston, J.S. (1995) Radiometal labeling of recombinant proteins by a genetically engineered minimal chelation site: technetium-99m coordination by single-chain Fv antibody fusion proteins through a C-terminal cysteinyl peptide. Proc. Natl. Acad. Sci., 92, 8358-62; Berndorff, D., Borkowski, S., Moosmayer, D., Viti, F., Mueller-Tiemann, B., Sieger, S., Friebe, M., Hilger C.S., Zardi, L, Neri, D., and Dinkelborg, L.M. (2006) Imaging of tumor angiogenesis using Tc-labeled human recombinant anti-ED-B fibronectin antibody fragments. J. Nucl. Med., 47, 1707- 1716), high pH (> 9.5) in the radiolabeling solution appeared to be a critical factor toward the success of the radiolabeling procedure. The ^99mTc products of these high- pH radiolabelings comprised a mixture of dimer and monomer forms, and the radiochemical yields in these reactions were taken as the total % of the two forms combined together. This was based on the assumption that the ^99mTc products comprised an equilibrium mixture of two forms of the same radiolabeled species. It was now unexpectedly found for Tc and/or Re radiolabeled scFv antibody fragments that monomer product generated at high radiolabeling pH is a degradation product, which cannot equilibrate to form the ^99mTc dimer product, and that the degradation product has inferior biological properties as compared to the dimer. Based on these results, we have concluded that ^99mTc monomer species should be considered undesirable impurities in ^99mTc radiolabelings of scFv antibody fragments, and furthermore that formulation studies should aim to minimize them.

Thus, it is an object of the present invention to provide stable and homogeneous compositions for scFv antibody fragments labelled with at least one radioactive isotope and uses of such compositions. In some embodiments, the radiochemical yield is optimized in such compositions while minimizing monomer impurities.

It should be noted that the compositions described herein can be derived from kits, which are typically comprised of one or more vials containing the critical components of the composition. When the kit vials are combined together along with optionally other non-kit components, the final composition results.

Isotopes, which can be used are for example Technetium, such as ^94mTc, ^99mTc, Rhenium, such as ¹⁸⁶Re, ¹⁸⁸Re, or other isotopes, such as ¹³¹ l, ¹²³l, ¹²⁴l, ^117mSn, ²⁰³Pb, ⁶⁶⁷⁷GGaa,, ⁶⁶⁸⁸GGaa,, ⁴⁴³³SScc,, ⁴⁴⁴⁴SScc,, ⁴⁴⁷⁷SScc,, ^{11 1100mm}llnn ^{11 1111} llnn,, ⁹⁷Ru, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹²¹Sn, ¹⁶¹Tb ¹⁵³Sm, ¹⁶⁶Ho, ¹⁰⁵Rh, ¹⁷⁷Lu, ⁷²As and ¹⁸F.

In a preferred embodiment, the radioactive Tc isotope is Tc or Tc. In another preferred embodiment the radioactive Re isotope is selected from ¹⁸⁸Re and ¹⁸⁶Re. In a particularly preferred embodiment, the radioactive Tc and/or Re isotope is Tc.

Preferably the isotopes are added as ions with the metal in the (VII) oxidation state. In particular, ^99mTc and ^94mTc as pertechnectate (Tc(VII)) or TcO₄ ^" and ¹⁸⁶Re and ¹⁸⁸Re as perrhenate (Re(VII)) or ReO₄ ^" are used. In another embodiment the isotopes are added as preformed Tc(V) or Re(V) oxo complexes of suitable exchange ligands.

The radioactively labelled scFv antibody fragments are preferably useful as agent for diagnosis of human or animal diseases, in particular of hyperproliferative diseases, like tumours. Such scFv antibody fragments preferably specifically bind to a target in a human or animal body which is indicative for a certain disease or disorder.

The radioactively labelled scFv antibody fragments are further preferably useful as agent for diagnosis of human or animal atherosclerosis.

In a preferred embodiment, the scFv specifically binds to a target selected from ED-B fibronectin, ED-A fibronectin, tenascin C (A1 splice isoform), PSMA, CEA, CD20, CD22, CD33, CD45, CD66, CD105, MUC1 , CA-125, HER2/neu, GRP78, alpha(v) integrin, gpA33, EGFR, TAG-72, G250, A33, Ep-CAM, CSA-p, gp38, L6 and SSTR.

In a particularly preferred embodiment, the scFv antibody fragment specifically binds to an extracellular matrix protein, in particular to ED-B fibronectin, ED-A fibronectin or Tenascin C isoform c. Most preferably, the scFv antibody fragment specifically binds to ED-B fibronectin.

A further embodiment of the invention relates to a composition for a radiolabeled scFv antibody fragment comprising at least one scFv antibody fragment and further components needed for radiolabeling, and a radioactive isotope, wherein the pH in the composition solution is 6.5 to 9.4.

In one embodiment, the invention relates to a composition for a radiolabeled scFv antibody fragment comprising at least one scFv antibody fragment, further

components needed for radiolabeling with a radioactive Tc and/or Re isotope, and at least one Tc and/or Re isotope, wherein the pH in composition solution is 6.5 to 9.4, preferably 8.5 to 9.2.

In a further embodiment, the invention relates to a composition, wherein the composition comprises at least one scFv antibody fragment comprising at least one chelator for Tc and/or Re, tin chloride, at least one buffer substance and optionally diluent and/or at least one exchange ligand, wherein the pH in the composition solution is 6.5 to 9.4, preferably 8.5 to 9.2. Beside the active ingredients, the inventive compositions also comprise

physiologically acceptable adjuvants, diluents and/ or carriers. Said adjuvants, diluents and carriers are well known to the skilled person and can easily be selected from the existing literature. In a further embodiment, the invention relates to a kit for radiolabeling a scFv antibody fragment comprising at least one vial wherein the kit comprises at least one scFv antibody fragment and further components needed for radiolabeling with a radioactive Tc and/or Re isotope, wherein the pH in the final radiolabeling reaction solution is in the range of 6.5 to 9.4, preferably 8.5 to 9.2

In one preferred embodiment, the kit comprises one vial.

In a further embodiment, the invention relates to a kit, wherein the kit comprises one vial, and wherein the one vial comprises at least one scFv antibody fragment comprising at least one chelator for Tc and/or Re, tin chloride, at least one buffer substance and at least one exchange ligand, wherein the pH in the the final radiolabeling reaction obtained after combining and incubating the contents of the vial with at least one radioactive Tc and/or Re isotope, and optionally a diluent is 6.5 to 9.4, preferably 8.5 to 9.2.

In another preferred embodiment, the kit comprises two vials.

In another embodiment, the present invention relates to a kit comprising at least one vial which comprises at least one scFv antibody fragment which comprises at least one chelator for Tc and/or Re (Vial 1 ), and at least a second vial comprising tin chloride, at least one buffer substance and optionally at least one exchange ligand (Vial 2), wherein the pH in the mixture obtained after combining and incubating Vial 1 , Vial 2, optionally a diluent and at least one radioactive Tc and/or Re isotope, is 6.5 to 9.4, preferably 8.5 to 9.2.

In a preferred embodiment the radioisotope added to a vial of the present kit invention is in the form of pertechnetate or perrhenate. Buffer substances, which can be used are for example Tris, Borax, Carbonate, Bicine, HEPES, TAPS (N-[tris(hyroxymethyl)methyl]-3-aminopropanesulfonic acid), MOPS, Phosphate, AMPD (2-amino-2-methyl-1 ,3-propanediol), TABS ( N-tris(hydroxyl- methyl)methyl-4-aminobutanesulfonic acid) and glycine.

For a higher pH range of 8.5 to 9.2, for example, Tris, Borax, Carbonate, Bicine, TAPS (N-[tris(hyroxymethyl)methyl]-3-aminopropanesulfonic acid), AMPD (2-amino- 2-methyl-1 ,3-propanediol), TABS ( N-tris(hydroxymethyl)-methyl-4-amino- butanesulfonic acid) and glycine can be used, and for a lower pH range of 6.5 to 8.2, for example, HEPES, MOPS and Phosphate can be used.

Preferably, the buffer substance is glycine.

Preferably, the amount of tin chloride in the composition or kit is about 20 to 200 g SnCI₂-2H₂O, more preferably about 40 to 125 g SnCI₂-2H₂O, most preferably about 60 ig SnCI₂-2H₂O.

The tin chloride, the at least one buffer substance and the at least one exchange ligand in the kit invention may be contained in one, two, three or more vials.

According to the present invention, "Vial 2" is understood as having all compounds in one vial or in two, three or more vials. Preferably, they are contained in one vial or in two vials, most preferred in one vial. For example the tin chloride (SnCI₂) of Vial 2 may be stored in one vial, and the exchange ligand and glycine in another vial.

The final pH of the kit is determined by pH meter or pH paper after combining all vial contents together with radionuclide solution and optionally additional diluent, for example water or saline.

In a preferred embodiment, the pH of the composition or kit is between 6.5 and 9.4, particularly preferred between 8.5 and 9.2.

In one embodiment, the present invention relates to a kit comprising

(a) a Vial 1 comprising an scFv antibody fragment which comprises a chelator for Tc and/or Re

(b) a Vial 2 comprising a an exchange ligand, glycine and tin chloride

wherein after combining Vial 1 , Vial 2 and at least one radioactive Tc and/or Re isotope, and incubating the mixture, the pH is ≤ 9.4, preferably ≤ 9.2.

Suitable exchange ligands are in particular tartrate salt, glucarate salt, gluconate salt, glucoheptonate salt, and edetate salt.

In a preferred embodiment, the exchange ligand is a tartrate salt, in particular L- tartaricacid, disodium dihydrate. In a preferred embodiment, the chelator for Tc and/or Re comprises at least one free thiol group.

A vial of the present kit invention may be for example stored as dry substance, solution, frozen solution or in lyophilized form. Preferably it is stored as solution, frozen solution or in lyophilized form, most preferably in lyophilized form.

In embodiments wherein the kit comprises at least two vials, Vial 1 may be for example stored as dry substance, solution, frozen solution or in lyophilized form. Preferably it is stored as solution, frozen solution or in lyophilized form, most preferred in lyophilized form.

In embodiments wherein the kit comprises at least two vials, Vial 2 may be for example stored as dry substance, solution, frozen solution or in lyophilized form. Preferably it is stored in lyophilized form. In case Vial 2 consists of several components, each of the components, independently of the others, may be stored as dry substance, solution, frozen solution or in lyophilized form.

In a preferred embodiment the scFv antibody fragment contains at least one tag, preferably one tag. In a preferred embodiment, the tag comprises at least one chelator, more preferably one chelator for the Tc and/or Re isotope, preferably for ^99mTc. In a preferred embodiment the chelator contains at least one free thiol group.

More preferably the chelator comprises at least one cysteine.

Most preferably the chelator is peptidic, comprising one cysteine plus two non- proline, naturally-occurring amino acids adjacent to the Cysteine on the N-terminal side, X-X-Cys. The chelator is preferably covalently attached to the scFv antibody fragment, especially preferably via a peptide linkage. It may be present at the C- terminus, and/or at the N-terminus and/or internally within the sequence of the protein. Preferably, the chelator is present at the C-terminus. When the peptidic chelator is present at the C-terminus or internally, the tag may consist of Cys alone or Cys plus an adjacent non-proline amino acid, X-Cys, with theremaining non-proline amino acids of the chelator (X-X or X, respectively) derived from the sequence of the scFv protein. A peptidic chelator at the N-terminus must include two adjacent amino acids, and therefore an N-terminal tag consists of at least 3 amino acids (X-X-Cys).

In a preferred embodiment, at least one vial of the kit in addition comprises EDTA. In a preferred embodiment, for embodiments wherein the kit comprises at least two vials, Vial 1 and/or Vial 2, more preferably Vial 2 in addition comprises EDTA.

In a further preferred embodiment, the composition or kit further comprises a Tc and/or Re isotope.

In a further embodiment, the present invention relates to a method of producing a composition comprising at least one scFv antibody fragment labelled with at least one radioactive Tc and/or Re isotope comprising the steps:

(a) combining the contents of the vials of a kit of the present invention, in case

more then one vial is present, and at least one radioactive Tc and/or Re isotope and (b) optionally a diluent, and

(c) incubation of the mixture, wherein after incubation, the pH is 6.5 to 9.4,

preferably 8.5 to 9.2. In a further embodiment, the present invention relates to a method of producing a composition comprising at least one scFv antibody fragment labelled with at least one radioactive Tc and/or Re isotope comprising the steps:

(a) combining the contents of Vials 1 and 2 and at least one radioactive Tc and/or

Re isotope and

(b) optionally a diluent, and

(c) incubation of the mixture, wherein after incubation, the pH is 6.5 to 9.4,

preferably 8.5 to 9.2.

The diluent is preferably an aqueous solution, preferably a saline solution, which may also be buffered. In particular, preferable diluents include saline or phosphate buffered saline. The diluent is in particular useful in case the vial components are in dry form, e.g. in lyophilized form, to help with combining the vial contents together.

The isotope is preferably selected from ^99mTc and/or ^94mTc and/or ¹⁸⁶Re and/or ¹⁸⁸Re, in particular it is ^99mTc.

In one embodiment, the incubation is performed at a temperature of about 5-45°C, preferably about 10-40°C, more preferably at about 15°-37°C most preferably at about 18°C to 25°C.

In a further embodiment, the incubation is performed for about 2 minutes to about several hours, like 1 , 2, 3, 4, 5, or 6 hours. Preferably, incubation is performed for a time of about 2 minutes to 60 minutes, more preferably for about 5 minutes to 45 minutes.

Optionally, the obtained solution may be subject to sterile filtration.

Optionally, the obtained solution may be subject to at least one purification step. Tc may be obtained by methods known by a skilled person, in particular by using a ^99mTc generator. The ^99mTc generator may be milked daily with saline to generate a solution containing ^99mTc pertechnetate. In a further embodiment, the present invention relates to a composition comprising at least one scFv antibody fragment labelled with at least one radioactive isotope obtainable by the method of the invention.

In a further embodiment, the present invention relates to a composition comprising at least one scFv antibody fragment labelled with at least one radioactive Tc and/or Re isotope obtainable by the method of the invention.

In a further embodiment, the present invention relates to a composition comprising at least one scFv antibody fragment labelled with at least one radioactive Tc and/or Re isotope, tartrate salt, glycine and tin chloride, having a pH ≤ 9.4, preferably≤9.2. In a further embodiment, the pH of the composition is preferably at least 8.0, more preferably at least 8.5.

A composition according to the present invention with 8.5≤ pH ≤9.2 is particularly preferred.

In a preferred embodiment, the composition in addition comprises EDTA. Preferably, the amount of Na₂EDTA-2H₂O in the composition and in the composition of the present invention is about 20 to 1000 g, more preferably between about 50 to 250ig, most preferably about 100 g.

In one embodiment, the scFv antibody fragment specifically binds to a target in a human or animal body which is indicative for a certain disease or disorder. In a preferred embodiment the scFv antibody fragment specifically binds to ED-B fibronectin

In a preferred embodiment, the scFv antibody fragment is L19 or a variant thereof having the same CDR sequences, in particular selected from AP38 and AP39. In a most preferred embodiment, the composition comprises Tc-AP39.

Preferably, the amount of AP39 in the composition and in the composition of the present invention is about 100 to 1000 g, more preferably between about 150 to 300 g, most preferably about 250 g.

The compositions of the present invention may be administered to a subject, e.g. a human patient or an animal. The diagnosis is then performed by a suitable detection method in particular SPECT, PET or scintigraphy.

The administration is preferably carried out by injecting the composition, which comprises the ^99mTc-labelled scFv antibody fragment, into a vein and/or artery of a patient to be examined and detecting the labeled diagnostic agent.

In a preferred embodiment, the scFv antibody fragment specifically binds to the ED- B-domain of fibronectin (FN). Antibodies and antibody fragments specifically binding to the ED-B-domain of FN are known in the prior art and are e.g. described in WO 97/45544.

In another embodiment, the scFv antibody fragment specifically binding to ED-B FN binds to a cryptic epitope. An example for such antibody is the BC-1 antibody.

Preferably, such scFv antibody fragments which bind to the ED-B domain of fibronectin exhibit a high affinity for the ED-B-domain of FN, in particular, the antibody binds to the ED-B fibronectin domain with nanomolar or subnanomolar affinity. Such antibodies and antibody fragments are known in the prior art and are e.g. described in WO99/58570. In a particularly preferred embodiment, the scFv fragments optionally have a peptide tag containing a chelator for a Tc and/or Re isotope, wherein the peptide tag comprises 1 to 50 amino acids, preferably 3 to 30, even more preferred 3 to 6 amino acids are used. Particularly preferred are tags comprising X-X-Cys, wherein "X" may be any naturally occurring amino acid except proline. In another embodiment, the tag is 1 or 2 amino acids in length located at the C-terminus or internally within the sequence of the protein, having the sequence Cys, or X-Cys, respectively. In this case 2 or 1 amino acids of the scFv itself serve as part of the chelator. Such tags are, suitable for chelating Tc and/or Re, preferably they are suitable for chelating ^99mTc, ^94mTc, ¹⁸⁶Re and/or ¹⁸⁸Re, in particular ^99mTc.

In particular preferred is the use of scFv fragments comprising the L19 scFv antibody fragment and variants thereof, which comprise the CDR sequences of the L19 scFv antibody fragment.

Particularly preferred is L19 scFv with a tag, wherein the antibody comprises as tag further amino acids, preferably at the C-terminus, which are capable of chelating Tc and/or Re. Such L19 scFv derivatives with a tag are disclosed in WO 03/055917. Most preferred is the use of AP38 and AP39, in particular AP39 disclosed in WO 03/055917.

In a further preferred embodiment, the scFv antibody fragments contains at least one CDR sequence of the L19 scFv antibody fragment.

In an especially preferred embodiment, the scFv antibody fragment comprises the CDR sequences of the L19 scFv antibody; in particular it comprises the sequences according to SEQ ID No. 14 to 19. In a further preferred embodiment, the scFv antibody fragment comprises the VL and VH chain of the L19 scFv antibody fragment. In a preferred embodiment, it comprises least one VH chain according to SEQ ID No. 01 or at least one VL chain according to SEQ ID No. 02. In an especially preferred embodiment, it comprises least one VH chain according to SEQ ID No. 01 and at least one VL chain according to SEQ ID No. 02.

In a further preferred embodiment, the scFv antibody fragment comprises the VH chain according to SEQ ID No. 01 and the VL chain according to SEQ ID No. 02. In a further preferred embodiment, the VH and the VL chains are connected by a peptide linker. The peptide linker preferably has a length of about 1 to 18 amino acids, more preferably of about 3 to 15 amino acids. In a preferred embodiment, the peptide linker comprises a sequence according to SEQ ID No. 03, or a sequence having at least 90% identity to the sequence according to SEQ ID No. 03.

In a most preferred embodiment, the VH chain according to Seq. ID No.1 is connected via a linker according to Seq. ID No. 3 to the VL chain according to Seq. ID No. 2 to form the scFv antibody according to Seq. ID No. 4. AP39 is preferably used in dimeric form, wherein the monomers of the dimer may be formed covalently or non-covalently, preferably non-covalently. In particular, AP38 according to SEQ ID No. 5 and AP39 according to SEQ ID No. 6 can be used.

Especially preferred as scFv antibody fragments are L19 scFv derivatives comprising (aa) at least one antigen binding site for the ED-B domain of fibronectin

comprising the complementarity-determining regions of the HCDR3 according to SEQ ID No. 16 and/or of the LCDR3 according to SEQ ID No. 19, or a variant thereof, which exhibits a deletion, insertion and/or substitution of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region, whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No.

4

(ab) at least one antigen binding site for the ED-B domain of fibronectin

comprising the complementarity-determining regions HCDR1 according to SEQ ID No. 14, HCDR2 according to SEQ ID No. 15, HCDR3 according to SEQ ID No. 16, LCDR1 according to SEQ ID No. 17, LCDR2 according to SEQ ID No. 18 and LCDR3 according to SEQ ID No. 19, or a variant thereof, which exhibits a deletion, insertion and/or substitution of up to 3 amino acids in the HCDR1 region, of up to 8 amino acids in the HCDR2 region, of up to 5 amino acids in the HCDR3 region, of up to 6 amino acids in the LCDR1 region, of up to 4 amino acids in the LCDR2 region and of up to 6 amino acids in the LCDR3 region, whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No. 4, or

(ac) at least one antigen binding site for the ED-B domain of fibronectin

comprising the sequence of the native L19 scFv, shown in SEQ ID No. 4, or a variation thereof, which exhibits a deletion, insertion and/or substitution of up to 30 amino acids, whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No. 4, and optionally

(ba) an amino acid sequence Xaai-Xaa₂-Xaa3-Cys shown in SEQ ID No. 7, whereby Xaai, Xaa₂, and Xaa3, independently of one another, represent a bond or any naturally occurring amino acid,

(bb) an amino acid sequence Xaai-Xaa₂-Xaa3-Cys-Xaa₄, shown in SEQ ID

No. 8 whereby Xaai, Xaa₂, Xaa3, and Xaa₄, independently of one another, represent a bond or any naturally occurring amino acid,

(be) an amino acid sequence (His)_n shown in SEQ ID No. 9, whereby n is an integer from 4 to 6, or

(bd) an amino acid sequence that comprises the sequence shown in SEQ ID

NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, whereby the C-terminus of

(aa), (ab), or (ac) is optionally bonded via a peptide bond to the N- terminus of (ba), (bb), (be) or (bd).

Preferably, Xaai , Xaa2, Xaa3, and Xaa4 do not represent proline.

Such molecules are disclosed in WO 03/055917.

Such scFv antibody fragments comprise an N-terminal antigen binding site for the extra-domain B (ED-B) of fibronectin selected from the antigen binding sites (aa), (ab) or (ac) and optionally a C-terminal amino acid sequence selected from the amino acid sequences (ba), (bb), (be) or (bd), whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No. 4.

In a preferred embodiment, the antigen binding sites for the extra-domain B (ED-B) of fibronectin of the labeled L19 derivative (aa) or (ab) comprise the complementarity- determining regions HCDR3 and/or LCDR3 or HCDR1 , HCDR2, HCDR3, LCDR1 , LCDR2 and LCDR3, shown in SEQ ID No. 14 to 19.

HCDRx according to the present invention means the Complementarity-determining region x the heavy antibody chain.

LCDRx according to the present invention means the complementarity-determining region x the light antibody chain. In addition to the complementarity-determining regions according to SEQ ID No. 14 - 19, the antigen binding sites for the extra-domain B (ED-B) of fibronectin of the labeled L19 derivative (aa) or (ab) can also comprise variants of these regions.

According to the invention, a variant of the HCDR1 region comprises a deletion, insertion and/or substitution of up to 3 amino acids in the HCDR1 region, i.e., a deletion, insertion and/or substitution of 1 , 2 or 3 amino acids relative to the sequence according to SEQ ID NO. 14. A variant of the HCDR2 region comprises a deletion, insertion and/or substitution of up to 8 amino acids in the HCDR2 region, i.e., a deletion, insertion and/or substitution of 1 , 2, 3, 4, 5, 6, 7 or 8 amino acids relative to the sequence according to SEQ ID NO. 15. Moreover, a variant of the HDCR3 region comprises a deletion, insertion and/or substitution of up to 5 amino acids in the HCDR3 region, i.e., a deletion, insertion and/or substitution of 1 , 2, 3, 4 or 5 amino acids relative to the sequence according to SEQ ID NO. 16. A variant of the LCDR1 region, however, comprises a deletion, insertion and/or substitution of up to 6 amino acids in the LCDR1 region, i.e., a deletion, insertion and/or substitution of 1 , 2, 3, 4, 5 or 6 amino acids relative to the sequence according to SEQ ID NO. 17. In addition, a variant of the LCDR2 region comprises a deletion, insertion and/or substitution of up to 4 amino acids in the LCDR2 region, i.e., a deletion, insertion and/or substitution of 1 , 2, 3 or 4 amino acids relative to the sequence according to SEQ ID NO. 18. A variant of the LCDR3 region comprises a deletion, insertion and/or substitution of up to 6 amino acids in the LCDR3 region, i.e., a deletion, insertion and/or substitution of 1 , 2, 3, 4, 5 or 6 amino acids relative to the sequence according to SEQ ID NO. 19.

According to this invention, the antigen binding site for the ED-B domain of fibronectin of the labeled L19 derivative (ac) comprises the sequence of native L19 scFv, shown in SEQ ID NO. 4, or a variation thereof, which exhibits a deletion, insertion and/or substitution of up to 30 amino acids, i.e., a deletion, insertion and/or substitution of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids relative to the sequence shown in SEQ ID NO. 4. The amino acid sequences (ba), (bb) or (be) of the labeled L19 derivative comprise the sequences Xaai-Xaa₂-Xaa3-Cys shown in SEQ ID No. 7, Xaai-Xaa₂-Xaa3-Cys-Xaa₄ shown in SEQ ID No. 8 or (His)_n shown in SEQ ID No. 9 whereby n is an integer from 4 to 6.

In a preferred embodiment of this invention, the amino acid sequence (ba) Xaar Xaa₂-Xaa3-Cys, shown SEQ ID No. 7 is the sequence Gly-Gly-Gly-Cys shown in SEQ ID No. 10 or Gly-Cys-Gly-Cys shown in SEQ ID No. 1 1 . Especially preferred is the sequence Gly-Gly-Gly-Cys shown in SEQ ID No. 10.

In another preferred embodiment of this invention, the amino acid sequence (bb) Xaai-Xaa₂-Xaa3-Cys-Xaa₄ shown in SEQ ID No. 8 is the sequence Gly-Gly-Gly-Cys- Ala shown in SEQ ID No. 12 or Gly-Cys-Gly-Cys-Ala shown in SEQ ID No. 13.

Especially preferred is the sequence Gly-Gly-Gly-Cys-Ala shown in SEQ ID No. 12.

In another preferred embodiment of this invention, the N-terminus of (aa), (ab) or (ac) is optionally connected via a peptide bond to the C-terminus of a linker amino acid sequence. The linker amino acid sequence preferably has a length of up to 30 amino acids, preferably up to 25 amino acids, and especially preferably up to 22 amino acids. Especially preferred is the linker amino acid sequence, which is the sequence shown in SEQ ID NO. 23.

Such linker amino acid sequences are disclosed in WO 2005/ 037312.

In WO 2009/036885, several labeled antibodies or antibody fragments against the ED-B domain are described as diagnostic reagents for stratifying a tumor.

According to this invention, especially preferred labeled L19 scFv antibody fragments comprise the sequences shown in SEQ ID NO. 4 (L19 scFv, native L19), SEQ ID NO. 5 (AP38), SEQ ID NO. 6 (AP39), L19(scFv)-GlyCysGlyCys, SEQ ID NO. 24,

L19(scFv)-GlyCysGlyCysAla, SEQ ID NO. 25 and SEQ ID NO. 26. Especially preferred is AP39, SEQ ID NO. 6. The tags allow chelation of ^99mTc and/or ^94mTc and/or ¹⁸⁶R and/or ¹⁸⁸Re. In a particularly preferred embodiment ^99mTc-labelled AP39 (^99mTc- AP39) is present in the composition.

The composition of the invention preferably comprising ^99mTc-labelled AP39 is preferably applied to the patient by parenteral or intravenous administration, more preferably by intravenous injection. The human dose is typically in the range of about 0.01 to 10 mg, preferably in the range of about 0.1 to 1 mg per patient.

The scFv antibody fragments are preferably produced recombinantly using methods known to the skilled person. In particular, prokaryotic, e.g. E. coli or eukaryotic expression systems, e.g. yeast or mammalian expression systems, can be used.

The diagnostic application of the compositions of the invention in vivo is particularly preferred.

In the most preferred embodiment, a composition ^99mTc-AP39 is used.

In a further embodiment, the present invention relates to the use of the compositions of the present inventions for diagnosis of a disease.

In a preferred embodiment, the disease is a hyperproliferative or inflammatory disease.

In a more preferred embodiment, the disease is a tumour.

The present invention further relates to a method of diagnosing a patient comprising

(a) administering a diagnostically effective amount of a composition of the

present invention to the patient

(b) performing diagnosis. Peptide linker is any linker, preferably a peptide linker, which is suitable for linking Vh and VI domains. Suitable linkers are for example described in Bird, et al, 1988;

Huston, et al, PNAS USA, 85, 5879-5883, 1988; EP 0 573 551 ; EP 0 623679 and EP 0 318554, which documents are introduced by reference.

"Specifically binding" or "specifically recognizing" as used herein refers to binding to the corresponding target. Typically, the binding molecule, antibody, antibody fragment or antibody mimetic binds with an affinity of at least about 1x10^"7 M, preferably of at least about 1 x10^"9 M, and binds to the predetermined target with an affinity that is at least two-fold greater than its affinity for binding to a non-specific target (e.g. BSA, casein) other than the predetermined target or a closely-related target. "Antibody" as used herein encompasses full length antibodies, comprising native antibodies, monoclonal antibodies, polyclonal antibodies and multispecific antibodies (e.g., bispecific antibodies), human antibodies, humanized antibodies, chimeric antibodies, and full IgG antibodies, as well as antibody fragments. The term "antibody fragment" refers to a portion of a full length antibody, in which a variable region or a functional capability is retained, namely the specific binding to the target. Examples of antibody fragments include, but are not limited to, a Fab, Fab', F(ab')2, Fd, Fv, scFv and scFv-Fc fragment, a diabody, a linear antibody, small immunoprotein formats, a single-chain antibody, a minibody, a diabody formed from antibody fragments, and multispecific antibodies formed from antibody fragments. Antibody fragments are usually smaller than full antibodies. Thereby, the

pharmacokinetics are different and some antibody fragments only consist of one polypeptide chain, which can make production easier. "Fv" is a minimum antibody fragment that contains a complete antigen-recognition and binding site consisting of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In this configuration, the three CDRs of each variable domain interact to define an antigen-binding site on the surface of the VH VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody.

A "single-chain Fv" or "scFv" antibody fragment is a single chain Fv variant comprising the VH and VL domains of an antibody, in which the domains are present in a single polypeptide chain and which is capable of recognizing and binding antigen. The scFv polypeptide optionally contains a polypeptide linker positioned between the VH and VL domains that enables the scFv to form a desired three- dimensional structure for antigen binding (see, e.g., Pluckthun, 1994, In The

Pharmacology of Monoclonal Antibodies, Vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315).

The VL domain associates non-covalently with the VH domain, whereas the CL domain is commonly covalently linked to the CH1 domain via a disulfide bond.

Particular amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia, et al., 1985). The term "hypervariable" refers to the fact that certain sequences within the variable domains differ extensively in sequence among antibodies and contain residues that are directly involved in the binding and specificity of each particular antibody for its specific antigenic

determinant. Hypervariability, both in the light chain and the heavy chain variable domains, is concentrated in three segments known as complementarity determining regions (CDRs) or hypervariable loops (HVLs). CDRs are defined by sequence comparison in Kabat, et al., 1991 , In: Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD., whereas HVLs are structurally defined according to the three-dimensional structure of the variable domain, as described by Chothia and Lesk, 1987, J. Mol. Biol. 196: 901 - 917.

Where these two methods result in slightly different identifications of a CDR, the structural definition is preferred. As defined by Kabat, CDR-L1 is positioned at about residues 24-34, CDR-L2, at about residues 50-56, and CDR-L3, at about residues 89-97 in the light chain variable domain; CDR-H1 is positioned at about residues 31 - 35, CDR-H2 at about residues 50-65, and CDR-H3 at about residues 95-102 in the heavy chain variable domain.

The term "label" refers to a detectable compound, moiety, or composition that is conjugated directly or indirectly to the antibody fragment.

Although fibronectins (FNs) are the product of the single FN gene, the resulting protein can exist in multiple forms which— apart from posttranslational modifications — arise from alternative splicing of its primary RNA transcript. This polymorphism which leads to as many as 20 different isoforms in human FN, thereby generating FNs with different solubility, cell adhesive and ligand-binding properties, provides cells with the possibility to modify the composition of the extracellular matrix (ECM) in a tissue-specific manner. Alternative splicing takes place in three regions of the primary RNA transcript: Exon usage or skipping leads to either inclusion or omission of two type-Ill repeats, extra-domain B (ED-B, also termed EIIIB or EDIII), which is inserted between FN type-Ill repeats III7 and III8, or/and extra-domain A (EDA, also termed EIIIA or EDI), inserted between FN type-Ill repeats 1111 1 and 11112. This type of splicing occurs in many vertebrates, including Xenopus, chicken, rat, dog and human. "ED-B" domain" is to be understood as the extra-domain B of human fibronectin. It is often referred to as ED-B, EIIIB or EDM.

Within the scope of this invention, the term "reduced form" means that the antibody fragment is present in monomeric or multimeric form wherein no detectable covalent linkage between a monomer and another monomer or a thiol-containing small molecule via S-S bonds is present. The reduced form of the scFv antibody fragment of the invention is preferably obtained by adding a suitable reducing agent. Suitable reducing agents are well known in the prior art and comprise for example TCEP (tris(2-carboxyethyl)phosphine) and 1 ,4-dimercapto-2,3-butanediols.

Description of the figures

Figure 1 shows formation of Monomer at pH 10.5 (37°C) as monitored by SE HPLC for cold AP39 (diamonds) and ^99mTc AP39 dimer (squares).

Figure 2 shows representative in vivo SPECT images from apoE^"'^" in comparison to WT mice 4h after intravenous administration of ^99mTc AP39 in coronary, sagital and transversal reconstruction. Furthermore, a corresponding overlay image from MRI and SPECT is depicted for apoE^"'^" and WT mice.

Figure 3 shows the quantification of the in vivo SPECT signal activity in apoE^"'^" and WT mice.

The following examples demonstrate the feasibility of the instant invention, however, not restricting the invention to those examples only.

Examples

Examplel : Preparation of AP39

AP39 was produced recombinantly by fermentation in Pichia Pastoris according to published procedures (Cunha, A.E., Clemente J.J., Gomes, R., Pinto, F., Thomaz, M., Miranda, S., Pinto, R., Moosmayer, D., Donner, P., and Carrondo, M.J.T. (2004) Methanol induction optimization for scFv antibody fragment production in Pichia pastoris. Biotechnol. and Bioeng., 86, 458-67) . During the preparative

chromatographic purification of the AP39 dimer product, monomer side product was also isolated. Example 2: Preparation of Vial 1 Frozen AP39

AP39 was reduced with tris-(2-carboxyethyl)phosphine hydrochloride (TCEP) according to previously published procedures (Berndorff, D., Borkowski, S.,

Moosmayer, D., Viti, F., Mueller-Tiemann, B., Sieger, S., Friebe, M., Hilger C.S., Zardi, L, Neri, D., and Dinkelborg, L.M. (2006) Imaging of tumor angiogenesis using ^99mTc-labeled human recombinant anti-ED-B fibronectin antibody fragments. J. Nucl. Med., 47, 1707-1716). The reduced AP39 was eluted from the NAP-5 size exclusion cartridge in 20 mM glycine buffer pH 8.5, diluted to 0.5 mg/mL in the same buffer, transferred into plastic tubes in 250 g (500 μΙ_) aliquots, and frozen. These reduced frozen samples of AP39 comprised Vial 1 of the composition for preparing the composition.

Example 3: Preparation of Vial 2 Lyophilized Stannous Tin

Small batches (50-100) of 5 ml_ serum vials containing lyophilized 2 mg L-tartaric acid, sodium salt dihydrate, 7.5 mg glycine, and 60 g stannous chloride dihydrate, pH adjusted to 9.2 with HCI or NaOH were prepared and stored at -20°C. During batching and dispensing the solution was sparged with argon to limit air oxidation of the stannous tin. These lyophilized vials comprised Vial 2 of the composition for preparing the composition. Example 4: Radiolabeling of AP39 with ^JJmTc and Quality Control.

To prepare ^99mTc AP39, Vial 1 (frozen AP39 solution) was thawed and warmed to room temperature, and the AP39 solution was transferred to a lyophilized AP39 composition Vial 2. ^99mTc pertechnetate generator eluate + saline totaling 1 .5 ml_ was added to the preparation vial (total preparation volume ¾ 2.0 ml_), and it was incubated at room temperature for 30 minutes.

In "wet kit" radiolabelings, the components of the Vial 2 composition were combined with Vial 1 as separate reagents. For example, a typical wet kit preparation was made as follows: the thawed contents of a Vial 1 AP39 vial was added to an empty 5 ml_ vial along with 2 mg disodium L-tartrate, glycine buffer pH 9.2 (200 μΙ_; 0.5 M), ^99mTc pertechnetate generator eluate (~ 20.2 mCi), and saline to make up the total volume to 2 mL. Finally, SnCl₂-2H₂O in ethanol freshly dissolved (60 g; 5 mg/mL) was added and the preparation was incubated at room temperature for 30 minutes.

The ^99mTc AP39 was analyzed by TLC on ITLC SG (Gelman) strips developed in aqueous saturated NaCI (SAS) and 5% sodium lauryl sulfate (SDS). The SDS TLC strips were pre-spotted with a drop of 5% HSA in water. The ITLC strips were spotted with 10-20 μί of radiolabeled product, developed in SAS or SDS, cut at R_f = 0.50 or 0.35, respectively, and the strip portions counted for radioactivity. The calculation for Radiochemical Purity (RCP) of ^99mTc-AP39 by TLC was: RCP = % Bottom SAS - % Bottom SDS

HPLC analysis of ^99mTc AP39 was done on a size-exclusion HPLC column (Toso Biosep TSK-gel G2000SWXL) with radiometric (γ) detection. The HPLC

radiochemical purity was determined as the % area of the main radiolabeled product peak.

TLC and HPLC radiochemical purity (RCP) results for 17 preparations are given in Table 1 . Table 1.

aBrackets indicate failing result. Passing results: TLC = RCP≥ 90%; HPLC = RCP > 85% with no single impurity > 5%. Example 5: Monomer Formation at pH 10.5.

A preparation of ^99mTc AP39 was prepared at pH 10.5 in a wet kit preparation as follows: reduced AP39 (100 μg), disodium L-tartrate (3.0 mg), 0.5 M glycine buffer pH 10.5 (200 μΐ), and 80 μg SnCl₂-2H₂O, were combined together in a 5 mL vial. ^99mTc pertechentate generator eluate (20 mCi) was added to the vial with saline to make the volume to 2 mL, and the reaction solution was heated at 37°C. The amount of ^99mTc AP39 monomer was monitored by SE HPLC with radiometric detection.

In a separate study to monitor AP39 protein stability at pH 10.5, reduced AP39 (500 g) was combined with the same ingredients as described above except without the ^99mTc pertechnetate. The amount of (cold) AP39 monomer was monitored by UV/vis HPLC. Monomer and Dimer peaks in HPLC chromatograms were integrated and %monomer was determined as (monomer area/monomer + dimer area) x100%. Results are shown in Figure 1 . Example 6: Radiolabeling of AP39 Monomer with ^99mTc

AP39 production monomer was reduced with TCEP as described above for the dimer, except the material was eluted into and diluted in saline. Radiolabeling was done using a wet kit preparation as described in Example 4. The following solutions were added to a plastic tube: reduced AP39 monomer in saline (100 g; 200 μΙ_), glycine buffer pH 8.6 (200 μΙ_; 0.5 M), ^99mTc pertechnetate generator eluate (150 μΙ_; 20.2 mCi), and SnCI₂-2H₂O in ethanol freshly dissolved (80 pg; 5 mg/mL). The reaction solution was incubated at 37°C for 30 minutes, and then purified on a NAP-5 Sephadex G25 cartridge eluted with saline. Combined NAP-5 purification fractions gave a sample containing 1 .95 mCi in 320 μΙ_. The direct labelled ^99mTc AP39 monomer sample had HPLC RCP = 87%.

Example 7: FPLC Isolation of ^99mTc AP39 Monomer and Dimer from a high-pH Incubation

Reduced AP39 was prepared at 0.5 mg/mL as described above in Example 2, except the material was eluted into and diluted in saline. A wet kit radiolabeling was conducted as described in Example 4 but at a high pH (10.5). The following solutions were added to a plastic tube: reduced AP39 in saline (100 g; 200 μί), glycine buffer pH 10.5 (200 μί; 0.5 M), ^99mTc pertechnetate generator eluate (172 μί; 18.5 mCi), and SnCl2-2H₂O in ethanol freshly dissolved (80 g; 5 mg/mL). The pH 10.5 reaction solution was incubated at 37°C for 2 hours. SE HPLC analysis of the reaction solution showed 77/23 ^99mTc dimer/monomer. The ^99mTc AP39 dimer and monomer products were isolated by FPLC and collected in -200 μί fractions (1 % PBS in water). Combined fractions of isolated ^99mTc AP39 dimer contained 1 .66 mCi in -0.8 mL; combined fractions of ^99mTc AP39 monomer contained 0.46 mCi in -1 .0 mL. The isolated ^99mTc AP39 dimer and monomer samples had HPLC RCP = 93% and 97%, respectively.

Example 8: Biodistribution Studies in Tumor Xenograft Mice

9^9mTc AP39 dimer and monomer samples were evaluated for biodistribution in F9 murine teratocarcinoma syngenic tumor nude mice. Nude mice (NMRI Nu/Nu, female, 24-35 g) were inoculated 1 1 -18 days before use with 1 x 10⁶ F9 tumor cells in the right hind limb. Mice were injected with approximately 1 .6 - 2.2 Ci of ^99mTc labeled scFv intravenously in the tail vein. Three mice per time point were sacrificed at 1 h, 3 h and 24 h p.i. for organ excision. Tissues collected included kidney, liver, lung, spleen, thyroid, heart, brain, bone (sample), muscle (sample), stomach, intestine (Gl), uterus, ovary, and blood (sample). Additionally, urine and feces were collected over time. The dissected tissues and collected excretions were counted for radioactivity in a gamma-counter (Compugamma LKB Wallac) and values of % ID/g and %ID were calculated and reported as mean value together with standard deviation.

The following ^99mTc samples were analyzed for biodistribution in tumor-bearing mice:

1 . ^99mTc AP39 Dimer direct labeled according to Example 4.

2. ^99mTc AP39 Monomer direct labeled according to Example 6.

3. ^99mTc AP39 Dimer from a high-pH incubation isolated by FPLC as described in Example 7.

4. ^99mTc AP39 Monomer from a high-pH incubation isolated by FPLC as

described in Example 7.

Biodistribution results are shown below in Table 2. Table 2.

include contents. Example 9: Effect of pH on radiolabeling of AP39 with ^aamTc.

Four wet kit radiolabelings (see Example 4), were prepared at various pHs as follows: Glycine buffer solutions 0.5 M were prepared at pH 8.6, 9.2, 9.8, and 10.5. Reduced AP39 in saline was prepared as described in Example 2 except the protein was eluted from the NAP column in saline. Reduced AP39 solution (100 g; 200 μί) was added to four empty 5 mL vials along with glycine buffer at the four different pH's (200 μΙ_; 0.5 M), 3 mg disodium L-tartrate, ^yymTc pertechnetate generator eluate (~ 20 mCi), and saline to make up the total volume to 2 mL. SnCl2-2H₂O in ethanol freshly dissolved (80 g; 5 mg/mL) was added and the preparation was incubated at 37°C for 30 minutes. All four radiolabellings were analyzed by SE HPLC as described in Example 4 initially and at 4 hours.

The HPLC results revealed 5 main radiolabeled species (in order of elution from the HPLC column): Tc-99m aggregate (larger impurity), Tc-99m AP39 dimer, Tc-99m AP39 monomer, Tc-99m TCEP (impurity), and Tc-99m pertechnetate (TcO₄ ^";

impurity). The radiolabeled TCEP impurity (-4-8%) results from residual TCEP reductant in the AP39 and can be eliminated by further purifying the AP39.

Therefore, the TCEP impurity was ignored in these studies. In other words, the results given in Table 3 correspond to the % area of the 4 other radiolabeled species peaks excluding the Tc-99m TCEP peak.

Table 3.

Example 10: Effect of incubation temperature on radiolabeling of AP39 with 9^9mTc.

Four wet kit radiolabelings (see Example 4), were prepared at pH 8.6 and incubated at various temperatures as follows: Reduced AP39 in saline was prepared as described in Example 2 except the protein was eluted from the NAP column in saline Reduced AP39 solution (100 g; 200 μί) was added to four empty 5 mL vials along with pH 8.6 glycine buffer (200 μΙ_; 0.5 M), 3 mg disodium L-tartrate, ^yymTc pertechnetate generator eluate (~ 20 mCi), and saline to make up the total volume to 2 mL. SnCl2-2H₂O in ethanol freshly dissolved (80 g; 5 mg/mL) was added and the preparation was incubated at room temperature (RT) (20 - 25°C), 37°C, 50°C, and 100°C for 30 minutes. All four radiolabellings were analyzed by SE HPLC as described in Example 4.

The HPLC results for the room temperature (RT), 37°C, and 50°C incubations are given in Table 4. The 100°C incubation preparation sample showed no activity recovered from the HPLC column (i.e. no discernable product or impurity). Results in Table 4 were calculated excluding the Tc-99m TCEP impurity as described in Example 9.

Table 4.

Example 11 : Effect of protein amount on radiolabeling of AP39 with Tc.

Three wet kit radiolabelings (see Example 4), were prepared at pH 8.6 with varying amounts of added AP39 protein as follows: Reduced AP39 in saline was prepared as described in Example 2 except the protein was eluted from the NAP column in saline. Reduced AP39 solution 0.5 mg/mL (100, 150, or 250 pg) was added to three empty 5 mL vials along with pH 8.6 glycine buffer (200 μί; 0.5 M), 3 mg disodium L- tartrate, ^99mTc pertechnetate generator eluate (~ 20 mCi), and saline to make up the total volume to 2 mL. SnCl₂-2H₂O in ethanol freshly dissolved (125 g; 5 mg/mL) was added and the preparation was incubated at room temperature for 30 minutes. All three radiolabellings were analyzed by SE HPLC as described in Example 4 at initial and 4 hour time points.

The HPLC results for the 100, 150, and 250 g AP39 preparations are given in Table 5. Results in Table 5 were calculated excluding the Tc-99m TCEP impurity as described in Example 9.

Table 5.

Example 12: Effect of stannous tin amount on radiolabeling of AP39 with

Two wet kit radiolabelings (see Example 4), were prepared at pH 8.6 with two levels of stannous tin as follows: Reduced AP39 in glycine buffer was prepared as described in Example 2. Reduced AP39 solution 0.5 mg/mL (250 Mg) was added to two empty 5 mL vials along with pH 8.6 glycine buffer (200 M 0.5 M), 3 mg disodium L-tartrate, ^99mTc pertechnetate generator eluate (~ 20 mCi), and saline to make up the total volume to 2 mL. SnCl2-2H₂O in ethanol freshly dissolved 5 mg/mL was added in two amounts (125 Mg and 60 Mg) and the preparation was incubated at room temperature for 30 minutes. Both radiolabelings were analyzed for RCP by TLC and SE HPLC as described in Example 4 at initial and 2 hour time points.

Results are given in Table 6.

Table 6.

All samples contain 3.4 - 6.9% Tc-99m TCEP by HPLC. Example 13: Targeted ED-B fibronectin SPECT imaging in experimental atherosclerosis in vivo

A "wet kit" ^99mTc AP39 preparation was made as described in Example 4 to evaluate its potential for imaging of atherosclerosis in 12 months old apoE^"'^" mice [C57BL/6 background; fed with western diet (0.21 % cholesterol; 20% fat, Altromin) since an age of six weeks] and in corresponding wild-type (WT) mice (C57BL/6; on normal chow). Four hours after intravenous administration of ^99mTc-anti-ED-B (148 MBq in 100 μΙ_) into apoE^{" _} (n=7) mice and WT mice (n=5), the animals were anesthetized by subcutaneous injection of rompun/ketamin (2:1 mL/kg). Imaging was performed using the 16-module variant of a Nucline Spirit DH-V SPECT camera (MEDISO, Budapest, Hungary) with a 10 Pinhole Collimator, HISPECT (SciVis GmbH, Germany).

Twenty-four hours before SPECT imaging all animals were imaged with an high resolution 7 Tesla MRI (Pharma- Scan^®70/16 AS, Bruker, Germany) using a T1 - weighted high resolution sequence, a midsystolic ECG and respiratory triggered 3D turbo-spin-echo sequence (RARE factor 2, Bruker). To get an anatomic correlation for the SPECT image, we overlayed the turbo-spin-echo MRI images obtained in the region of aortic root which is identified by the cusps of the aortic valves [Dietrich T, Hucko T, Bourayou R, Jahnke C, Paetsch I, Atrott K, Stawowy P, Grafe M, Klein C, Schnackenburg B, Fleck E, Graf K (2009) High resolution magnetic resonance imaging in atherosclerotic mice treated with ezetimibe. Int J Cardiovasc Imaging] with the SPECT images.

In vivo SPECT imaging of apoE^"'^" mice demonstrated a significant signal activity in the thoracic area which co-localized with the aortic arch and the supra-aortic arteries as shown in Fig.2. The activity was significantly increased in apoE^"'^" compared to WT mice (apoE^"'^": 52.23 ± 40.64 (1/1 )^*(ccm) vs. 9.46 ± 4.97 (1/1 )^*(ccm) in WT mice, meaniSD;) [Fig.3]. Increased activities were also observed in the gall bladder, the liver, and the kidneys in apoE^"'^" and WT mice [Fig.2].

Claims

What is claimed is:

1 . A composition for a radiolabeled scFv antibody fragment comprising at least one scFv antibody fragment, further components needed for radiolabeling, and a radioactive isotope wherein the pH in the composition solution is in the range of 6.5 to 9.4.

A composition according to claim 1 , wherein the radioactive isotope is selected from the isotope ¹³¹ l, ¹²³l, ¹²⁴l, ^117mSn, ²⁰³Pb, ⁶⁷Ga, ⁶⁸Ga, ⁴³Sc, ⁴⁴Sc, ⁴⁷Sc, ^{1 10m}ln 1^{1 1} ln, ⁹⁷Ru, ⁶⁴Cu, ⁶⁷Cu, ⁸⁶Y, ⁸⁸Y, ⁹⁰Y, ¹²¹Sn, ¹⁶¹Tb ¹⁵³Sm, ¹⁶⁶Ho, ¹⁰⁵Rh, ¹⁷⁷Lu, ⁷²As and ¹⁸F.

A composition for a radiolabeled scFv antibody fragment comprising at least one scFv antibody fragment, further components needed for radiolabeling with a radioactive Tc and/or Re isotope, and at least one radioactive Tc and/or Re isotope, wherein the pH in the composition solution is in the range of 6.5 to 9.4, preferably in the range of 8.5 to 9.2.

A composition according to claim 1 to 3, wherein the composition comprises at least one scFv antibody fragment, at least one chelator for Tc and/or Re, tin chloride, at least one buffer substance, at least one radioactive Tc and/or Re isotope, and optionally a diluent and/or at least one exchange ligand, wherein the pH in the composition solution is in the range of 6.5 to 9.4

A composition according to any of claims 1 -4, wherein the pH is in the range of 8.5 to 9.2.

A composition according to any of claims 1 -5 wherein the scFv antibody fragment is an scFv antibody fragment which contains at least one tag suitable for chelating at least one radioactive Tc and/or Re isotope.

7. A composition according to any of any of claims 3 to 6, wherein the radioactive Tc and/or Re isotope is selected from ^99mTc, ^94mTc, ¹⁸⁶Re and ¹⁸⁸Re.

8. A composition according to any of claims 4 to 7, wherein the exchange ligand is tartrate.

9. A composition according to any of claims 4 to 8, wherein the buffer substance is glycine.

10. A composition according to any of claims 4 to 9, wherein the composition in

addition comprises EDTA.

1 1 .A kit for radiolabeling a scFv antibody fragment comprising at least one vial

wherein the kit comprises at least one scFv antibody fragment and further components needed for radiolabeling with a radioactive Tc and/or Re isotope, wherein the pH in the final radiolabeling reaction solution is in the range of 6.5 to 9.4, preferably 8.5 to 9.2.

12. A kit according to claim 1 1 , wherein the kit comprises one vial, and wherein the one vial comprises at least one scFv antibody fragment comprising at least one chelator for Tc and/or Re, tin chloride, at least one buffer substance and at least one exchange ligand, wherein the pH in the final radiolabeling reaction obtained after combining and incubating the contents of the vial with at least one radioactive Tc and/or Re isotope, and optionally a diluent is in the range of 6.5 to 9.4

13. A kit according to claim 1 1 , wherein the kit comprises at least one vial comprising at least one scFv antibody fragment which comprises at least one chelator for Tc and/or Re (Vial 1 ), and at least a second vial comprising tin chloride, at least one buffer substance and optionally at least one exchange ligand (Vial 2), wherein the pH in the mixture obtained after combining and incubating Vial 1 , Vial 2, optionally a diluent and at least one radioactive Tc and/or Re isotope, is in the range of 6.5 to 9.4.

14. A kit according to any of claims 1 1 -12, wherein the pH is in the range of 8.5 to 9.2.

15. A kit according to claims 1 1 -14 in which the radioisotope added to the kit vial is in the form of pertechnetate or perrhenate.

16. A kit according to any of claims 1 1 -15 wherein the scFv antibody fragment is an ScFv antibody fragment which contains at least one tag suitable for chelating at least one radioactive Tc and/or Re isotope.

17. A kit according to any of any of claims 1 1 to 16, wherein the radioactive Tc and/or Re isotope is selected from ^99mTc, ^94mTc, ¹⁸⁶Re and ¹⁸⁸Re.

18. A kit according to any of claims 12 to 17, wherein the exchange ligand is tartrate.

19. A kit according to any of claims 12 to 18, wherein the buffer substance is glycine.

20. A kit according to any of claims 1 1 to 19, wherein the kit in addition comprises EDTA.

21 .A composition according to any of claims 1 - 10, characterized in that the scFv antibody fragments are L19 scFv derivatives, which comprise

(aa) at least one antigen binding site for the ED-B domain of fibronectin

comprising the complementarity-determining regions of the HCDR3 according to SEQ ID No. 16 and/or of the LCDR3 according to SEQ ID No. 19, or a variant thereof, which exhibits a deletion, insertion and/or substitution of up to 5 amino acids in the HCDR3 region and up to 6 amino acids in the LCDR3 region, whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No. 4,

(ab) at least one antigen binding site for the ED-B domain of fibronectin

comprising the complementarity-determining regions HCDR1 according to SEQ ID No. 14, HCDR2 according to SEQ IDNo. 15, HCDR3 according to SEQ ID No. 16, LCDR1 according to SEQ ID No. 17, LCDR2 according to SEQ ID No. 18 and LCDR3 according to SEQ ID No. 19, or a variant thereof, which exhibits a deletion, insertion and/or substitution of up to 3 amino acids in the HCDR1 region, of up to 8 amino acids in the HCDR2 region, of up to 5 amino acids in the HCDR3 region, of up to 6 amino acids in the LCDR1 region, of up to 4 amino acids in the LCDR2 region and of up to 6 amino acids in the LCDR3 region, whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No. 4, or

at least one antigen binding site for the ED-B domain of fibronectin comprising the sequence of the native L19 scFv, shown in SEQ ID No. 4, or a variation thereof, which exhibits a deletion, insertion and/or substitution of up to 30 amino acids, whereby the antigen binding site exhibits the same function as the native L19 scFv shown in SEQ ID No. 4, and optionally

an amino acid sequence Xaai-Xaa₂-Xaa3-Cys shown in SEQ ID No. 7, whereby Xaai, Xaa₂, and Xaa3, independently of one another, represent a bond or any naturally occurring amino acid,

an amino acid sequence Xaai-Xaa₂-Xaa3-Cys-Xaa₄, shown in SEQ ID No. 8 whereby Xaai, Xaa₂, Xaa3, and Xaa₄, independently of one another, represent a bond or any naturally occurring amino acid, an amino acid sequence (His)_n shown in SEQ ID No. 9, whereby n is an integer from 4 to 6, or

an amino acid sequence that comprises the sequence shown in SEQ ID NO. 20, SEQ ID NO. 21 or SEQ ID NO. 22, whereby the C-terminus of (aa), (ab), or (ac) is optionally bonded via a peptide bond to the N- terminus of (ba), (bb), (be) or (bd).

22. A composition according to claim 21 , characterized in that the L19 scFv

derivatives comprise SEQ ID NO. 4 (L19 scFv, native L19), SEQ ID NO. 5

(AP38), SEQ ID NO. 6 (AP39), L19(scFv)-GlyCysGlyCys, SEQ ID NO. 24, L19(scFv)-GlyCysGlyCysAla, SEQ ID NO. 25 or SEQ ID NO. 26.

23. A process for the production of a composition comprising at least one scFv

antibody fragment labelled with at least one radioactive Tc and/ or Re isotope comprising the following steps:

a) combining the contents of all kit vials according to any of claims 12 to 22 and at least one radioactive Tc and/ or Re isotope, and optionally a diluent ,

b) incubation of the mixture, wherein after incubation the pH is in the

range of 6.5 to 9.4.

24. A composition comprising at least one scFv antibody fragment labelled with at least one radioactive Tc and/or Re isotope obtainable by the process according to claim 23.

25. A composition comprising at least one scFv antibody fragment labelled with at least one radioactive Tc and/or Re isotope, tartrate salt, glycine and tin chloride, having a pH in the range of 6.5 to 9.4.

26. A composition according to claim 24 or 25, which in addition comprises EDTA.

27. A composition according to any of claims 24 to 26 wherein the scFv antibody

fragment specifically binds to ED-B fibronectin

28. A composition according to claim 27 wherein the scFv antibody fragment is L19 scFv or a variant thereof having the same CDR sequences, in particular selected from AP38 and AP39.

29. A composition according to any of claims 24 to 28 wherein the isotope is ^99mTc.

30. Use of a composition according to claim 1 to 10 and 21 to 29 as agent for

diagnosis of human or animal diseases.

31 . Use of a kit according to claim 1 1 to 20 as agent for diagnosis of human or animal diseases.

32. Use according to claim 30 and 31 , wherein the disease is a hyperproliferative disease.

33. Use according to claim 30 to 32, wherein the disease is a tumour or is

atherosclerosis.