The present invention relates to stabilized radiopharmaceutical compositions, its use and process for the production.

Especially, the present invention relates to stabilized organic molecules, proteins and peptides, which are radiolabeled.

More specific, the invention relates to compositions which comprise a

radiolabeled antibody and a stabilizer. In recent years, the emphasis in nuclear medicine has shifted toward targeted molecular imaging and therapy. In this approach, localized targets such as antigens, receptors, enzymes, or pathological phenotypes are targeted by radioactive isotopes to identify or treat a disease. Because the abundance of these molecular targets is often low, it is imperative to have very specific radiopharmaceuticals with high purity. The specificity of the radiopharmaceutical is largely determined by its ability to bind to its target. Hence, targeting molecules with high target binding affinities like antibodies, antibody fragments, peptides, and receptor ligands are often incorporated into radiopharmaceuticals. A number of radionuclides are routinely employed in nuclear medicine, including imaging isotopes such as ^99m Tc, ¹¹¹ In, ¹⁸ F, ²⁰¹ TI, ¹²³ l, ¹³¹ 1, ⁸² Rb and therapeutic isotopes such as ¹³¹ 1, ⁸⁶ Sr, ¹⁷⁷ Lu, ¹⁵³ Sm. Radionuclides are typically attached to targeting molecules in a radiolabeling process using common chemical reactions. For example, radiometals usually employ chelation / complexation chemistry for radiolabeling the targeting molecule, and radiohalogens usually utilize

nucleophilic substitution chemistry.

Stability is also critical to the success of a targeted molecular

radiopharmaceutical. The final drug product from the radiolabeling process needs to have an adequate shelf life to ensure it does not degrade substantially prior to injection into the patient. A long shelf life is important for radiolabeled products that are made at a central location and then shipped to the hospital for use. Radiopharmaceuticals are especially prone to degradation because the radiation emitted by the radionuclide can break chemical bonds in the targeting molecule or in other components in the radiopharmaceutical composition, thus causing autoradiolysis. Autoradiolysis is a particular problem for radiotherapeutic radiopharmaceuticals because they contain more damaging, higher-energy nuclides such as β-emitters (e.g. ¹³¹ 1, ¹⁸⁸ Re, ⁹⁰ Y) and a-emitters (e.g. ²¹³ Bi, ²¹² Bi, ²¹¹ At, ²²⁵ Ac, ²²³ Ra), and because the radiotherapeutic patient dose typically has higher activity and higher specific activity. Autoradiolysis is also a more serious problem for radiopharmaceuticals comprising more complex targeting molecules (e.g. biomolecules, proteins and peptides), since these contain more key chemical bonds that can be broken to elicit critical radiation damage.

Thus, stabilizers are often employed in radiopharmaceutical compositions to counteract the effects of autoradiolysis and maximize the drug substance stability. Such stabilizers must be non-toxic and must maintain the product's radiochemical purity for the duration of its shelf life. In addition, an acceptable radiopharmaceutical stabilizer must not interfere with delivery of the radionuclide to the target site.

Thus, an object of the instant invention is to make radiopharmaceuticals available, which are stabilized and thus have a longer shelf life and do not degrade substantially prior to injection into the patient. In US 5,384, 1 13, US 4,233,284 and WO2009/059977 methods for stabilizing radiopharmaceuticals by adding gentisates are described.

In US20090005595 the use of gentisic acid or gentisate salts for stabilization of ¹²³ l radiopharmaceuticals is described.

In US 6,685,912 and US 5,679,318 the stabilization of radiopharmaceuticals using ascorbic acid is described. In US 7,351 ,397 and US 6,881 ,396 stabilized radiopharmaceutical compositions have been disclosed containing hydrophilic 6-hydroxy chroman stabilizers.

In US 7,351 ,398 and US 6,902,718 stabilized radiopharmaceutical compositions have been disclosed containing hydrophilic thioether stabilizers.

In US 6,174,513 radiopharmaceutical compositions are described , which are stabilized by the addition of surfactants.

In US20050063902 stabilized radiopharmaceutical compositions comprising an amino-substituted aromatic carboxylic acid and a diphosphonic acid in

combination are described.

Other compounds such as ethyl alcohol, benzyl alcohol, inositol, and HSA have been used to stabilize radiopharmaceutical compositions. Furthermore, compounds such as gluthation, (poly) phenolic antioxidants, vitamin A, pycnogenol, lycopens, coenzyme Q10, quercetin, epicatechin, gallic acid , sodiumbisulfite sodium sulfite, hydrogensulfite, butylhydroxyanisol,

butyl hydroxytoluol NDGA and the like, have been used to stabilize parenteral protein formulations.

[ ¹³¹ l] L19-SIP is a radiolabeled antibody fragment targeting ED-B fibronectin that has been proposed as a radiotherapeutic drug for treatment of solid human tumors (D. Berndorff, S. Borkowski, S. Sieger, A. Rother, M. Friebe, F. Vita, C.S. Hilger, J.E. Cyr, and L.M. Dinkelborg, (2005) Radioimmunotherapy of solid tumors by targeting ED-B fibronectin: Identifiaction of the best-suited

radioimmunoconjugate. Clin. Cancer Res., 1 1 (19 Suppl), 7058s). As a protein radiotherapeutic, [1-131 ] L19-SIP is susceptible to autoradiolysis, and a formulation / composition containing suitable stabilizers is needed.

Sodium Iodide ¹³¹ 1 is a radiopharmaceutical for diagnosis and treatment of thyroid cancer or overactive thyroid. It is typically administered to the patient orally as a radioactive capsule. The capsules are often prepared at the site of use from an aqueous solution of [ ¹³¹ l] sodium iodide. The radioiodide solution itself is also an approved radiopharmaceutical which can be given by intravenous injection. In considering possible autoradiolytic processes in the [ ¹³¹ l] sodium iodide solution, iodide is a simple elemental anion, and therefore possesses no chemical bonds to break. However, the iodide is prone to oxidation by air or oxidizing free radicals to generate volatile free radioiodine [ ¹³¹ l] l ₂ . Therefore, the antioxidant / reductant thiosulfate has been employed as a reducing agent stabilizer for [ ¹³¹ l] sodium iodide solutions.

Thiosulfate has also been administered directly to human patients; it is used as an i.v. antidote in high concentrations for intoxications with cyanides (e.g. 25% solutions). In WO00/52031 , thiosulfate was given as a possible component in

radiopharmaceutical kits for radiolabeling chelator-conjugated proteins or peptides with therapeutic radiometal isotopes. It was noted as one of several (16) possibilities of radioprotectants that could be included in a formulation buffer vial. However, it was not specified as part of a preferred embodiment, and no results were given demonstrating the efficacy of thiosulfate for this purpose.

In JP60048937, conditions are described for radiolabeling a protein with radioactive iodide containing a reducing agent such as thiosulfate using a method employing an oxidant such as chloramine-T. An optimum ratio of reductant to oxidant is specified. However, this concerns the process for labeling proteins and is not related to stabilizing a radiopharmaceutical medicament, with regard to a longer shelf life and less degradation prior to injection into a patient.

Moreover, none of the above mentioned state of the art discloses stabilized compositions, which comprise thiosulfate together with more complex

radiopharmaceuticals, such as labeled organic molecules, labeled proteins and labeled peptides that have a longer shelf life and less degradation prior to injection into a patient.

Especially, nothing is known with regard to stabilized radiolabeled protein, antibody, or antibody fragment compositions together with thiosulfate that have a longer shelf life and less degradation prior to injection into a patient.

It is well known to a skilled person that a radiopharmaceutical can be understood as a radioactive isotope or a compound that comprises an organic molecule, a protein, a peptide and/or an antibody, which is labeled with a radioactive isotope. In principle, all radioactive isotopes can be used within the radiopharmaceutical. Preferred radioactive isotopes that can be used are, for example ²¹³ Bi, ²¹² Bi, ²¹¹ At, ²²⁵ Ac, ^94m Tc, ^99m Tc, ¹⁸⁶ Re, ¹⁸⁸ Re, ¹³¹ l, ¹²³ l, ¹²⁴ l, ^117m Sn, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m ln, ¹¹¹ ln, ⁸² Rb, ⁹⁷ Ru, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb

¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As, ¹⁸ F, ⁷⁶ Br, ⁸⁶ Sr, ²²³ Ra, ²⁰¹ TI.

The radiopharmaceutical can comprise one or more of the same or different of these isotopes.

It has now surprisingly been found that the addition of thiosulfate and/ or ascorbate, respectively the sodium salts thereof to formulations of more compl radiopharmaceuticals possessing chemical bonds susceptible to radiolytic cleavage can significantly increase the product's shelf life and give less degradation prior to injection into a patient. In addition, it has now surprisingly been found that the addition of thiosulfate, ascorbate, ascorbic acid, gentisate, gentisic acid, methionine, maltose, inositol, human serum albumin, benzyl alcohol, trehalose, povidone, niacinamide, and/ or cysteine to formulations of the complex protein radiopharmaceutical [ ¹³¹ l] L19- SIP can significantly increase the product's shelf life and give less degradation prior to injection into a patient. Moreover, thiosulfate and ascorbate are unexpectedly particularly useful for stabilizing radiohalide products, like [ ¹³¹ l] L19-SIP. The term "radioprotectant" stands for an ingredient in a radiopharmacetical composition that inhibits radiolytic damage or degradation of the

radiopharmaceutical drug substance or of components of the drug product. In the context of this invention, radioprotectants include thiosulfate, ascorbate, ascorbic acid, gentisate, gentisic acid, methionine, maltose, inositol, human serum albumin, benzyl alcohol, trehalose, povidone, niacinamide, cysteine, and the alkali and earth alkali salts of thiosulfate, ascorbate and gentisate, such as for example lithium-, sodium-, potassium-, rubidium-, caesium-, beryllium-, magnesium-, calcium-, strontium- and barium-thiosulfate, ascorbate and gentisate.

Especially preferred radioprotectants include lithium-, sodium-, potassium-, rubidium-, caesium-, beryllium-, magnesium-, calcium-, strontium- and barium- thiosulfate or -ascorbate. The inventive stabilized composition can comprise one or more of the above mentioned radioprotectants.

The most preferred radioprotectant is sodium-thiosulfate and sodium-ascorbate. The inventive stabilized composition may preferably comprise sodium-thiosulfate and sodium-ascorbate, alone or in combination.

The term "radiopharmaceutical" means a radiolabeled organic molecule, a radiolabeled protein or a radiolabeled peptide. Especially it means a radiolabeled antibody fragment, for example the radiolabeled antibody fragment L19-SIP (Seq. ID No. 1 and Seq. ID No. 2). The organic molecules, proteins and peptides, especially antibodies can be radiolabeled with one or more of the same or different isotopes selected from

213 _B . 212 _B . 211 _At> 225^ 94^ 9^ 186^ 188 _{Re >} 131 ^ 123^ 124^ H Zmg^ 203 _{pb >}

⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m ln, ¹¹¹ ln, ⁸² Rb, ⁹⁷ Ru, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As, ¹⁸ F, ⁷⁶ Br, ⁸⁶ Sr, ²²³ Ra and/or ²⁰¹ TI.

Preferred are those radiopharmaceuticals which are labeled with a radiohalogen.

Such radiohalogens are, for example, those of the radio isotopes ¹³¹ 1, ¹²³ l, ¹²⁴ l, ⁷⁶ Br, and ¹⁸ F.

Most preferred is the radiohalogen ¹³¹ 1, and the most preferred

radiopharmaceutical is ¹³ 1-L19-SIP. While ¹³¹ 1-L19-SIP can be combined with all of the above mentioned

radioprotectants, sodium-thiosulfate and -ascorbate are the most preferred radioprotectants.

Most preferred is a composition which comprises the radiopharmaceutical ¹³¹ 1- L19-SIP and one or more of the radioprotectants thiosulfate, ascorbate, ascorbic acid, gentisate, gentisic acid, methionine, maltose, inositol, benzyl alcohol, trehalose povidone, niacinamide, and cysteine.

A selected composition comprises ¹³¹ 1-L19-SIP as radiopharmaceutical and sodium-thiosulfate and/ or -ascorbate/ascorbic acid as radioprotectant.

The preferred concentration of sodium-ascorbate/ ascorbic acid in the ³¹ 1-L19- SIP composition is 25-100 mg/mL, more preferred is 50-75 mg/mL, most preferred is 75 mg/mL. The preferred concentration of sodium thiosulfate in the ¹³ 1-L19-SIP composition is 25-100 mg/mL, more preferred is 50-75 mg/mL, most preferred is 50 mg/mL.

The inventive compositions can be used as a medicament for diagnosis or treatment of a disease, especially hyperproliferative or inflammatory diseases, such as cancer.

The inventive compositions, which are stable ready to use radiopharmaceutical compositions can be produced according to the following steps:

a) radiolabeling an organic molecule, a protein or a peptide in solution with an isotope, selected from ²¹³ Bi, ²¹² Bi, ²¹¹ At, ²²⁵ Ac, ^94m Tc, ^99m Tc, ¹⁸⁶ Re, 1 ⁸⁸ Re, ¹³¹ l, ¹²³ l, ¹²⁴ l, ^117m Sn, ²⁰³ Pb, ⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m ln, 1 ¹¹ ln, ⁸² Rb, ⁹⁷ Ru, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, ⁹⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb ¹⁵³ Sm, ¹⁶⁶ Ho, 1 ⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As, ¹⁸ F, ⁷⁶ Br, ⁸⁶ Sr, ²²³ Ra and/ or ²⁰¹ TI,

b) optionally purifying the radiolabeled product to get a pure compound, c) adding one or more radioprotectants to the solution, selected from

thiosulfate, ascorbate, ascorbic acid, gentisate, gentisic acid, methionine, maltose, inositol, human serum albumin, benzyl alcohol, trehalose, povidone, niacinamide, cysteine and/or lithium-, sodium-, potassium-, rubidium-, caesium-, beryllium-, magnesium-, calcium-, strontium- and barium-thiosulfate, -ascorbate and/or -gentisate to the organic molecule, protein or peptide, to form the composition,

d) optionally diluting the product solution to obtain the ready to use

composition,

e) optionally dividing the composition into clinical doses, and finally f) optionally freezing the clinical doses prior to frozen shipment to the clinical site.

Especially, the inventive compositions comprise radiolabeled proteins that can be produced according to the following steps: a) radiolabeling a protein in solution with an isotope, selected from ²¹³ Bi,

212 _B . 211 _At 225 _ACi 94^ 99^ 186^ 188 _{Re >} 131 ^ 123^ 124^ H Zmg^ 203 _{p b >}

⁶⁷ Ga, ⁶⁸ Ga, ⁴³ Sc, ⁴⁴ Sc, ⁴⁷ Sc, ^110m ln, ¹¹¹ ln, ⁸² Rb, ⁹⁷ Ru, ⁶⁴ Cu, ⁶⁷ Cu, ⁸⁶ Y, ⁸⁸ Y, 9 ⁰ Y, ¹²¹ Sn, ¹⁶¹ Tb ¹⁵³ Sm, ¹⁶⁶ Ho, ¹⁰⁵ Rh, ¹⁷⁷ Lu, ⁷² As, ¹⁸ F, ⁷⁶ Br, ⁸⁶ Sr, ²²³ Ra and/ or ²⁰¹ TI,

b) optionally purifying the radiolabeled protein to get a pure compound, c) adding one or more radioprotectants to the solution, selected from

thiosulfate, ascorbate, ascorbic acid, gentisate, gentisic acid, methionine, maltose, inositol, human serum albumin, benzyl alcohol, trehalose, povidone, niacinamide, cysteine and/or lithium-, sodium-, potassium-, rubidium-, caesium-, beryllium-, magnesium-, calcium-, strontium- and barium-thiosulfate, -ascorbate and/or -gentisate to the organic molecule, protein or peptide, to form the composition,

d) optionally diluting the product solution to obtain the ready to use

composition,

e) optionally dividing the composition into clinical doses, and finally f) optionally freezing the clinical doses prior to frozen shipment to the clinical site. Especially, the protein under step a) is L19-SIP and the isotope is ¹³¹ 1, the purification method under step b) is size exclusion or anion exchange

chromatography, and the radioprotectant under step c) is thiosulfate or thiosulfate salts, and/ or is ascorbate or ascorbate salts, as mentioned above, most preferred is sodium-thiosulfate and/ or sodium-ascorbate.

A typical clinical dose as used in a patient having a bodyweight of about 70 kg the daily dose as administered is of 200-300 mCi ¹³¹ 1-L19-SIP for radiotherapy and 4-8 mg of L19-SIP protein. The inventive composition can be used for parenteral administration, such as intravenous, intramuscular, subcutaneaous or intratumoural administration, to warm-blooded animals, especially to humans. The composition comprises the active ingredient alone or, together with a pharmaceutically acceptable carrier. The dosage of the active ingredient depends upon the disease to be treated and upon the species, gender, age, weight, and individual condition, the individual pharma-cokinetic data, and the mode of administration.

The inventive composition can also used as a method for the prophylactic or especially therapeutic management of the human or animal body, to a process for the preparation thereof (especially for the treatment of tumours) and to a method of treating tumour diseases, especially those mentioned hereinabove.

The inventive composition can be used for the prophylactic or especially therapeutic management of neoplastic and other proliferative diseases of a warm-blooded animal, especially a human or a commercially useful mammal requiring such treatment, especially suffering from such a disease.

Preference is given to the use of solutions of the active ingredient, especially isotonic aqueous solutions. The pharmaceutical composition may be sterilized and/or may comprise excipients other than the radioprotectants described herein, for example preservatives, stabilizers, wetting agents and/or emulsifiers, solubilizers, salts for regulating osmotic pressure and/or buffers and are prepared in a manner known per se, for example by means of conventional dissolving and lyophilizing processes. The said solutions may comprise viscosity-increasing agents, typically sodium carboxymethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, or gelatins, or also solubilizers, for example Tween 80 [polyoxyethylene(20)sorbitan mono-oleate; trademark of ICI Americas, Inc. USA].

The manufacture of injectable composition is usually carried out under sterile conditions, as is filling, for example into ampoules or vials, and the sealing of the containers.

For parenteral administration, aqueous solutions of an active ingredient in water- soluble form, for example of a water-soluble salt, that contain viscosity-increasing substances, for example sodium carboxymethylcellulose, sorbitol and/or dextran, and, if desired, stabilizers, are especially suitable.

Solutions such as are used, for example, for parenteral administration can also be employed as infusion solutions.

As used herein, cancer includes hematologic malignancy such as acute leukemia, malignant lymphoma, multiple myeloma and macroglobulinemia as well as solid tumors such as colon cancer, cerebral tumor, head and neck tumor, breast carcinoma, pulmonary cancer, esophageal cancer, gastric cancer, hepatic cancer, gallbladder cancer, bile duct cancer, pancreatic cancer, nesidioblastoma, renal cell carcinoma, adrenocortical cancer, urinary bladder carcinoma, prostatic cancer, testicular tumor, ovarian carcinoma, uterine cancer, chorionic carcinoma, thyroid cancer, malignant carcinoid tumor, skin cancer, malignant melanoma, osteogenic sarcoma, soft tissue sarcoma, neuroblastoma, Wilms tumor and retinoblastoma.

The pharmaceutical composition may be prepared with generally used diluents or excipients such as filler, extender, binder, moisturizing agent, disintegrator, surfactant and lubricant. The pharmaceutical composition may have a variety of dosage forms depending on its therapeutic purpose.

Suitable carriers and adjuvants may be such as recommended for pharmacy, cosmetics and related fields in: Ullmann's Encyclopedia of Technical Chemistry, Vol. 4, (1953), pp. 1 -39; Journal of Pharmaceutical Sciences, Vol. 52 (1963), p. 918ff; H.v.Czetsch-Lindenwald, "Hilfsstoffe fur Pharmazie und angrenzende Gebiete"; Pharm. Ind. 2, 1961 , p.72ff; Dr. HP. Fiedler, Lexikon der Hilfsstoffe fur Pharmazie, Kosmetik und angrenzende Gebiete, Cantor KG, Aulendorf in Wurttemberg, 1971.

Further pharmacologically effective adjuvants and carriers are, for example, described in Remington's Pharmaceutical Science, 15* ⁿ ed. Mack Publishing Company, Easton Pennsylvania (1980), which is hereby incorporated by reference.

For preparing injection, the solution is sterilized and preferably isotonic with blood. It may be prepared using diluents commonly used in the art; for example, water, ethanol, macrogol, propylene glycol, ethoxylated isostearyl alcohol, polyoxyisostearyl alcohol and polyoxyethylene sorbitan fatty acid esters. The pharmaceutical preparation may contain sodium chloride necessary to prepare an isotonic solution, glucose or glycerin, as well as usual solubilizers, buffers and soothing agents.

An administration route of the pharmaceutical combination is not limited, and selected depending on patient's age, sex, severity of disease and other conditions. For example, injection may be intravenously administered solely or in combination with a common infusion fluid such as glucose, amino acids and the like, or if necessary, intramuscularly, subcutaneously, intratumorally or intraperitoneally as a sole preparation.

The dose of the pharmaceutical combination of this invention may be selected, depending on their dosage form, patient's age, sex and severity of disease, and other conditions, as appropriate.

For parenteral applications, such as intravenous application a composition has a typical pH range of pH 3 to 9, preferably a range of pH 5 to 8, and most preferred a pH range of 6 to 7.5. The osmolality for such intravenous application to a human patient is in the range of 100 to 1500 mOsmol/ kg, preferably in the range of 200 to 800 mOsmol/ kg and most preferred in the range of 250 to 400 mOsmol/ kg. A further parameter for the inventive composition is the temperature range for storage conditions and shelf life. The temperature range normally is 8 to 35°C, especially 15 to 30°C, and most preferred 21 to 25°C.

Description of the figure

Fig. 1 describes the sequence of the L19 SIP homodimer, sequence monomer 1 , as well as sequence monomer 2.

The following examples demonstrate the process for the production and the use of the instant invention, however not restricting the invention to those examples only. Examples

Example 1

Synthesis of L19-SIP The small immunoprotein (SIP) format of the single chain antibody fragment L19 was made recombinantly according to published procedures (Borsi L, Balza E., Bestagno M., Castellani P., Carnemolla B., Biro A., Leprini A., Sepulveda J., Burrone O., Neri D., and Zardi L. (2002) Selective targeting of tumoral vasculature: comparison of different formats of an antibody (L19) to the ED-B domain of fibronectin. Int. J. Cancer, 102, 75-85).

The L19 SIP homodimer comprises the two L19 SIP monomers of sequence monomer 1 (Sequence ID No. 1 ) and sequence monomer 2 (Sequence ID No. 2). Both monomers are linked by disulfide bridge Cys 357 - Cys 357 to form the 714 amino acids long L19 SIP homodimer, having a molecular mass of 77053 Da.

Further disulfide bridges can exist at positions Cys 22 - Cys 96, Cys 151 - Cys 217 and/or Cys 268 - Cys 328

Labelling of L19 SIP with 131 -lodine:

L19 SIP is labelled with 131 -iodine using the chloramine-T method which leads to a covalent incorporation of ¹³¹ 1 into tyrosyl residues of the protein. At this point, it is not known which tyrosine residue(s) in the L19-SIP protein is(are)

radioidinated. The molecular mass of ¹³¹ 1- L19SIP is 77053 + (131 )n Da . Example 2

Quality control of ¹³¹ l L19-SIP

Immunoreactivity assay (antigen binding)

ED-B fibronectin antigen protein was made recombinantly according to published procedures (Carnemola B., Neri D., Castellani P., Leprini A., Neri G., Pini A., Winter G., and Zardi L. (1996) phage antibodies with pan-species recognition of the oncofetal angiogenesis marker fibronectin ED-B domain. Int. J. Cancer, 68, 397-405). The antigen protein was bound to CNBr activated sepharose 4B according to standard procedures. Briefly, the activated sepharose resin was washed with 1 mM HCI and mixed with ED-B protein at a ratio of 4 mg protein per mL of resin in buffer A (100 mM NaHCOV 500 mM NaCI pH 8.3). The suspension was incubated overnight at 4°C, and the resin was washed subsequently with buffer A to remove unbound ED-B protein. Residual activated groups were blocked with 1 M ethanolamine pH 8.0, and the resin was finally stored in PBS.

ED-B resin (50 μΙ_) prepared as described as described above was loaded into a Pasteur pipet (column) and equilibrated with 1 .5 mL of dilute PBS (approximately 0.05 mM phosphate pH 7.4. ¹³¹ 1 L19-SIP sample solution (200 μΙ_) was loaded onto the resin column and the loading flow-through volume collected (FT). The column was washed with 1 .5 mL of dilute PBS collecting the wash eluate (W), and finally the product was eluted from the resin with 1 .5 mL of 100 mM triethylamine solution (E). The eluted column (C), load flow-through (FT), wash (W), and elution (E) were counted in a dose calibrator for radioactivity.

Immunoreactivity was determined as the percentage of total activity binding to the ED-resin:

IR = (C + E) /(C + E + W + FT) x 100% Determination of % Radioiodide by TLC

A small volume (< 10μΙ_) of ¹³¹ 1 L19-SIP sample was spotted at the origin of a 10 cm silica gel 60 (Merck) TLC plate, and the plate was developed in 85:15 methanokwater mobile phase (-7.5 cm run length). Developed TLC strips were analyzed for radioactivity profile on a radio-TLC scanner. Radiochromatogram peaks were integrated and the % radioiodide was determined as the percentage of total reactivity retained at the solvent front. Purity by SE HPLC

¹³¹ 1 L19-SIP (20 μί) sample solution was injected onto a size exclusion analytical HPLC column (Tosoh TSK-gel G2000SWXL, 300 x 7.8 mm) with TSK buffer mobile phase (0.1 M Na ₂ HP0 ₄ / 0.1 M Na ₂ S0 ₄ / 0.05% NaN ₃ ; pH 6.7) in an isocratic 30 minute elution at 0.5 mL/min flow rate with radiometric detection. The main product peak eluted at -16-18 minutes. Radiochromatogram peaks were integrated and the purity was determined as the percentage of main product peak area relative to the total area of all peaks.

Example 3

Excipient Stability Study A

¹³¹ l L19-SIP was prepared as follows: L19-SIP protein (4 mg in 1 1 mL), [1-131 ] sodium iodide solution (196 mCi in 5.8 mL) and phosphate buffer (0.5 mL of 0.05 mM pH 7.4) were added together and mixed. The radiolabeling reaction was initiated by addition of 17.4 mg of chloramines-T oxidant solution (0.30 mL of 58 mg/mL solution) and incubated for 3 minutes with further mixing. The product was purified promptly using a size exclusion Sephadex G25 fine column (GE Amersham Hiprep). The reaction solution was loaded onto the column and the column was eluted with 20 mL of dilute PBS. The 20 mL fraction corresponding to 10 ml to 30 mL eluted from the column was collected as the product solution. The total recovered product activity is 158 mCi. HPLC purity was 91 .6%, whereas the purity by TLC was 98.1 %. Immunoreactivity was 94.3%.

In order to test the stabilizing effect of several added excipients at various concentrations, the purified sample of ¹³¹ 1 L19-SIP described above was divided into 17 aliquots (7.9 mCi; 1 .35 mL) and these were combined with excipients to make stability test samples as described below.

Methionine at 5, 10, and 25 mg/mL

Inositol at 5, 10, 25, and 50 mg/mL

Maltose at 5, 10, 25, and 50 mg/mL

Sodium thiosulfate at 5, 10, 25, and 50 mg/mL

Trolox at 5 mg/mL

The inositol, maltose, and sodium thiosulfate were added as 10x concentrated solutions in saline, while the methionine and trolox samples were added directly as solids. A control sample was also generated by combining an 1-131 L19-SIP aliquot with an equivalent volume (0.15 mL) of saline. All samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 3, 5, and 7 days. Results are given in Tables 1 , 2, and 3. Table 1 : HPLC Purity Results (%). Stability Study A

Concentration Day 0 Day 1 Day 3 Day 5 Day 7

Methionine 5 mg/mL 86.7 84.2 68.4 64.2 57.7

10 mg/mL 87.5 86.1 68.2 68.1 56.5

25 mg/mL 86.7 88.5 69.8 69.2 61.8

Maltose 5 mg/mL 85.2 81.2 63.3 52.4 42.0

10 mg/mL 83.3 82.9 60.6 55.2 43.0

25 mg/mL 86.4 84.9 57.3 57.2 45.4

50 mg/mL 86.1 80.9 59.9 55.9 47.7

Inositol 5 mg/mL 86.9 83.8 57.1 59.6 50.7

10 mg/mL 86.5 83.2 62.8 64.8 53.9

25 mg/mL 87.7 84.8 64.5 61.9 54.8

50 mg/mL 87.3 83.8 64.2 63.9 53.2

Thiosulfate 5 mg/mL 92.5 90.9 85.1 80.8 73.6

10 mg/mL 90.8 90.9 84.9 81.7 77.4

25 mg/mL 94.5 94.0 91.3 87.4 74.6

50 mg/mL 91.8 92.6 94.2 90.8 86.6

Trolox 5 mg/mL 85.2 79.3 68.6 68.5 0.0

Control - 91.6 83.4 29.3 23.8 19.8

Table 2: Immunoreactivity Results (%). Stability Study A

Concentration Day 0 Day 1 Day 3 Day 5 Day 7

Methionine 5 mg/mL 96.7 88.0 72.9 62.6 61.0

10 mg/mL 96.6 88.1 75.7 64.2 54.8

25 mg/mL 97.2 89.6 79.6 48.6 58.9

Maltose 5 mg/mL 89.1 84.4 66.3 57.0 50.0

10 mg/mL 92.8 85.4 65.2 57.2 53.7

25 mg/mL 93.6 86.4 66.0 57.7 53.6

50 mg/mL 89.7 85.9 66.2 55.6 50.7

Inositol 5 mg/mL 94.8 87.1 64.6 56.2 56.2

10 mg/mL 93.4 89.5 70.4 58.9 60.7

25 mg/mL 94.1 88.7 68.5 59.9 62.8

50 mg/mL 92.3 89.3 71.6 63.7 61.9

Thiosulfate 5 mg/mL 97.3 94.4 83.5 79.4 70.0

10 mg/mL 96.2 94.7 88.5 81.3 78.0

25 mg/mL 96.4 95.0 92.3 87.9 78.5

50 mg/mL 95.8 96.9 91.7 91.5 88.2

Trolox 5 mg/mL 94.6 84.8 77.6 65.8 52.9

Control - 94.3 81.6 59.8 47.6 43.7

Table 3: Radioiodide Results (%). Stability Study A

Concentration Day 0 Day 1 Day 3 Day 5 Day 7

Methionine 5 mg/mL 1.8 7.9 20.3 26.2 34.2

10 mg/mL 3.7 8.4 19.6 25.5 34.9

25 mg/mL 3.0 7.1 17.4 21.8 29.3

Maltose 5 mg/mL 1.5 10.1 20.6 26.0 40.8

10 mg/mL 2.2 14.9 24.1 31 .8 42.3

25 mg/mL 1.4 8.1 24.0 30.5 41.1

50 mg/mL 2.4 9.3 25.6 31.5 41.2

Inositol 5 mg/mL 3.2 9.6 15.6 22.7 40.1

10 mg/mL 2.0 7.5 21.7 21.6 39.2

25 mg/mL 3.8 10.4 20.6 24.3 34.7

50 mg/mL 0.9 9.7 21.6 28.0 36.2

Thiosulfate 5 mg/mL 0.0 3.4 10.2 15.6 24.6

10 mg/mL 1.1 6.3 8.6 13.6 19.7

25 mg/mL 0.0 5.4 7.2 10.5 1 1.9

50 mg/mL 0.0 3.3 5.5 10.6 8.7

Trolox 5 mg/mL 0.0 7.9 19.1 21.3 45.2

Control - 1.9 6.6 23.8 25.6 41.7

Example 4

Excipient Stability Studies B1 -B3

I L19-SIP was prepared in 3 separate preparations B1 , B2, and B3 by the following general procedure: L19-SIP protein (0.5 mg in 1 .2 mL), [ ¹³¹ l] sodium iodide solution (10 mCi in 10 μΙ_) and phosphate buffered saline (0.5-1 .1 mL of 0.05 mM pH 7.4) were added together and mixed. The radiolabeling reaction was initiated by addition of 1 .1 - 1 .3 mg of chloramine-T oxidant solution (200 μί) and incubated for 3 minutes. The product was purified promptly using a small size exclusion Sephadex G25 fine column (GE Amersham PD10) equilibrated in 10 mM PBS. The reaction solution was loaded onto the column and the column was eluted with 8.5-8.9 mL of dilute PBS and fractions were collected. Fractions containing the main activity were combined; the product eluted after 1 .0-1 .5 mL in a combined fraction volume of 1 .7-3.3 mL. The total recovered product activity was 5.09-9.42 mCi (yield was 51 -94%) with a concentration of 2.27-2.82 mCi/mL. Quality control results for the 3 preparations are given in Table 4.

Table 4: QC results for ¹³¹ l L19-SIP preparations B1 , B2, and B3. HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) results (%)

In study B1 , the stabilizing effects of 4 different excipients (maltose, methionine, gentisic acid, and ascorbic acid) at -10 mg/mL concentration vs. a no-excipient control were evaluated. Excipient solutions -30 mg/mL were made up in PBS and adjusted to pH 7.4 with NaOH if necessary. Test samples were made by combining 200 μί of excipient solution with 400 μί of ¹³¹ 1 L19-SIP solution. A control sample was generated by combining an ¹³¹ 1 L19-SIP aliquot with an equivalent volume (200 μΙ_) of PBS. All B1 samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 2, 3, and 6 days. Results are given in Table 5.

Table 5: HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) Results (%). Stability Study B1. Effect of maltose, methionine, gentisic acid, and ascorbic acid at 10 mg/mL

In study B2, the stabilizing effects of 4 different excipients (trehalose, cysteine, benzyl alcohol, and sodium thiosulfate) at -10 mg/mL concentration vs. a no- excipient control were evaluated. Excipient solutions -30 mg/mL were made up in PBS and adjusted to pH 7.4 with NaOH if necessary. Test samples were made by combining 200 μί of excipient solution with 400 μί of ¹³¹ 1 L19-SIP solution. A control sample was generated by combining an ¹³¹ 1 L19-SIP aliquot with an equivalent volume (200 μΙ_) of PBS. All B2 samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 2, 4, and 6 days. Results are given in Table 6.

Table 6: HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) Results (%). Stability Study B2. Effect of trehalose, cysteine, benzyl alcohol, and thiosulfate at 10 mg/mL

Cysteine excipient sample exhibited precipitated activity at day 4.

In study B3, the stabilizing effects of 3 different excipients (thioglycerol, inositol, and human serum albumin HSA) at -10 mg/mL concentration and 1 excipient at 25 mg/mL concentration (ascorbic acid) vs. a no-excipient control were evaluated. Concentrated (3x) excipient solutions (-30 or -75 mg/mL) were made up in PBS and adjusted to pH 7.4 with NaOH if necessary. Test samples were made by combining 200 μΙ_ of excipient solution with 400 μΙ_ of ¹³¹ 1 L19-SIP solution. A control sample was generated by combining an ¹³¹ 1 L19-SIP aliquot with an equivalent volume (200 μΙ_) of PBS. All B3 samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 2, 3, 5, and 7 days. Results are given in Table 7.

Table 7: HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) Results (%). Stability Study B3. Effect of thioglycerol, inositol, and HSA at 10 mg/mL, and ascorbic acid at 25 mg/mL

Day 0 Day 1 Day 2 Day 3 Day 5 Day 7

Thioglycerol HPLC 94.2 91 .3 65.0 53.7 55.6 52.5 (10 mg/mL) IR 80.1 60.7 58.4 59.5 46.8 47.1

TLC 1 .9 1 .9 2.4 1 .9 2.0 2.1

Inositol HPLC 95.2 94.9 90.7 87.7 85.2 79.3 (10 mg/mL) IR 59.5 77.1 68.8 74.8 61 .2 61 .9

TLC 2.6 2.9 3.5 7.2 8.2 8.2

HSA HPLC 93.5 87.1 79.8 78.2 71 .6 65.3

(10 mg/mL) IR 76.3 78.0 59.9 60.6 53.0 39.8

TLC 2.8 3.0 4.3 2.2 4.6 2.5

Ascorbic acid HPLC 96.2 94.8 92.9 92.9 89.3 86.9 (25 mg/mL) IR 74.1 78.0 75.0 78.7 64.7 55.3

TLC 0.9 2.9 3.2 2.9 4.8 3.6

Control HPLC 95.0 92.9 89.0 87.4 71 .6 65.1

IR 74.6 73.2 71 .3 68.3 56.4 28.2

TLC 2.8 7.8 6.9 6.6 10.4 12.4 Example 5

Excipient Stability Studies C1 -C2

I L19-SIP was prepared in 2 separate preparations C1 and C2 by the following general procedure: [ ¹³¹ l] sodium iodide solution (140-152 mCi in 20 μΙ_) in a small vial was transferred to a 10 mL reaction vial containing L19-SIP protein (3.1 -3.2 mg in 2.4-2.5 mL) by rinsing the iodide vial and transfer lines with 1 .5 mL of phosphate buffered saline (PBS, 100 mM). The radiolabeling reaction was initiated by transferring 1 mg of chloramine-T oxidant solution in water (250 μί) to the reaction vial and subsequently rinsing the chloramine-T transfer lines with 0.25 mL of 100 mM PBS. The reaction was incubated for 1 .5 minutes. The product was purified promptly by pumping the reaction solution onto a size exclusion Sephadex G25 fine column (Hiprep; GE Amersham) equilibrated in 10 mM PBS, and subsequently eluting the column with 60 mL of 100 mM PBS. The elution of main product was monitored by an in-line radiation detector, and a product fraction corresponding to ~5 mL to -13 mL of column eluation volume was collected. The total recovered product activity for reaction C1 was 96 mCi (yield 69%) in 6.65 mL (14.5 mCi/mL). The total recovered product activity for reaction C2 was 1 12 mCi (yield 74%) in 8.3 mL (13.5 mCi/mL). Additional 100 mM PBS (~3 mL) was added to the product fractions to get a stock ¹³¹ 1 L19-SIP sample with 10 mCi/mL activity concentration.

In study C1 , the stabilizing effects of thiosulfate excipient at varying

concentrations as well as thiosulfate in combination with another excipient (gentisate, ascorbate, or methionine) vs. a no-excipient control were evaluated. In particular, the 7 test samples included:

1 . Sodium thiosulfate (10 mg/mL)

2. Sodium thiosulfate (25 mg/mL)

3. Sodium thiosulfate (50 mg/mL)

4. Sodium thiosulfate (75 mg/mL)

5. Sodium thiosulfate (50 mg/mL) + gentisic acid (10 mg/mL) 6. Sodium thiosulfate (50 mg/mL) + ascorbic acid (25 mg/mL)

7. Sodium thiosulfate (50 mg/mL) + methionine (10 mg/mL)

Excipient solutions at 3 x concentration were made up in PBS and adjusted to pH 7.4 with NaOH if necessary. Test samples were made by combining 0.5 mL of excipient solution with 1 .0 mL of stock ¹³¹ 1 L19-SIP solution. A control sample was generated by combining an ¹³¹ 1 L19-SIP aliquot with an equivalent volume (0.5 mL) of PBS. All C1 samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 3, 5, and 7 days. Results are given in Table 8.

Table 8: HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) Results (%). Stability Study C1. Effect of sodium thiosulfate at 10, 25, 50, and 75 mg/mL; and 50 mg/mL thiosulfate in combination with 10 mg/mL gentisic acid (GA), 25 mg/mL ascorbic acid (AA), or 10 mg/mL methionine (Met)

In study C2, the stabilizing effects of ascorbate excipient at varying

concentrations, thiosulfate at 50 mg/mL, povidone at 10 mg/mL, and thiosulfate in combination with niacinamide vs. a no-excipient control were evaluated. In particular, the 7 test samples included:

1. Ascorbic acid (10 mg/mL)

2. Ascorbic acid (25 mg/mL)

3. Ascorbic acid (50 mg/mL)

4. Ascorbic acid (75 mg/mL)

5. Sodium thiosulfate (50 mg/mL)

6. Povidone (10 mg/mL)

7. Sodium thiosulfate (50 mg/mL) + niacinamide (10 mg/mL)

Test samples containing excipients and a control sample were made as described above for experiment C1. All C2 samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 4, 5, and 7 days. Results are given in Table 9. Table 9: HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) Results (%). Stability Study C2. Effect of ascorbic acid (AA) at 10, 25, 50, and 75 mg/mL, sodium thiosulfate at 50 mg/mL, povidone at 10 mg/mL, and 50 mg/mL thiosulfate in combination with 10 mg/mL niacinamide (NiA)

Example 6

Excipient Stability Studies D1 -D2

¹³¹ 1 L19-SIP was prepared in 2 separate preparations D1 and D2 by the procedure described in Example 5 except starting with slightly higher amounts of [131 1] sodium iodide solution (D1 = 161 mCi; D2 = 164 mCi; in 30 μΙ_) and L19- SIP protein (3.6 mg; D1 in 2.6 mL; D2 in 2.7 mL).

In study D1 , the stability of a high-activity (-1 10 mCi) sample of 1-131 L19-SIP containing 75 mg/mL ascorbic acid was compared to an equivalent sample with lower-activity (-10 mCi). The total recovered product activity for reaction D1 was 120 mCi (yield 75%) in 10.2 mL (1 1 .8 mCi/mL). Additional 100 mM PBS (1 .8 mL) was added to the product fraction to get a stock ¹³¹ 1 L19-SIP sample with 10 mCi/mL activity concentration. 5.99 mL of ascorbic acid solution at 3 x

concentration (450 mg/mL) was added to the 1-131 L19-SIP stock solution to generate a product sample with 75 mg/mL ascorbic acid. 10 mCi (1 .5 mL) of this solution was removed and added to a separate vial as the low-activity Sample 1 . The remainder of the product solution (-1 10 mCi) comprised the high-activity Sample 2. The two D1 samples were tested for HPLC purity, immunoreactivity, and TLC purity at 0, 1 , 3, 5, and 7 days. Results are given in Table 10.

In study D2, the stability of a high-activity (-124 mCi) sample of 1-131 L19-SIP containing 50 mg/mL sodium thiosulfate was compared to an equivalent sample with lower-activity (-10 mCi). The total recovered product activity for reaction D2 was 134 mCi (yield 82%) in 10.6 mL (12.6 mCi/mL). Additional 100 mM PBS (2.8 mL) was added to the product fraction to get a stock ¹³¹ 1 L19-SIP sample with 10 mCi/mL activity concentration. 6.69 mL of sodium thiosulfate solution at 3 x concentration (150 mg/mL) was added to the ¹³¹ 1 L19-SIP stock solution to generate a product sample with 50 mg/mL sodium thiosulfate. 10 mCi (1 .5 mL) of this solution was removed and added to a separate vial as the low-activity

Sample 1 . The remainder of the product solution (-124 mCi) comprised the high- activity Sample 2. The two D2 samples were tested for HPLC purity,

immunoreactivity, and TLC purity at 0, 1 , 3, 5, and 7 days. Results are given in Table 10. Table 10: HPLC Purity (HPLC), Immunoreactivity (IR), and TLC Radioiodide (TLC) Results (%). Stability Studies D1 and D2. D1 : effect of ascorbic acid (AA) at 75 mg/mL in low activity (10 mCi) vs. high activity (110 mCi) samples. D2: effect of sodium thiosulfate at 50 mg/mL in low activity (10 mCi) vs. high activity (124 mCi) samples

Example 7

Excipient Stability Studies of the cold protein

L19-SIP protein was received in PBS at a concentration of 0.35mg/ml. The Protein solution were concentrated using a Vivacell 250 concentration cell (10000 MWCO). After concentration, the solution was filtered using a 0.22μιη PVDF filter. The resulting concentration was 2.6mg/ml. The protein solution was then transferred into the Soerensen buffer pH 6.5 using an Aekta Explorer and an AxiChrom column. The concentration of the final solution of L19 SIP in the Soerensen buffer was 1 .51 mg/ml and a second concentration step was applied to the protein using the above mentioned set up resulting in a 2.03 mg/ml protein solution. The protein concentration was determined at 280nm using a Nanodrop 2000 (Thermo Scientific). Excipient (Na-Thiosulfate and Na-Ascorbate) solutions were prepared in Soerensen buffer and added to the Protein solutions to result in the final concentrations given in table 1 1 .

After combination of the protein solution with the excipient solution, the resulting solutions were again filtered using a 0.22μιη PVDF filter.

Table 11 : Composition of L19-SIP excipient solutions

Formulation Excipients Concentration Concentration

of of

Radioprotective Polysorbate

Excipients in 80

the Formulation

F01 Blank (L19 in 0 0%

Soerensen

buffer)

F02 Na- 75 mg/ml 0%

Ascorbate

F03 Na- 150 mg/ml 0%

Ascorbate

F04 Thiosulfate 50 mg/ml 0%

F05 Thiosulfate 100 mg/ml 0% Formulation Excipients Concentration Concentration

of of

Radioprotective Polysorbate

Excipients in 80

the Formulation

F01 .1 Blank (L19 in 0 0.01 %

Soerensen

buffer)

F02.1 Na- 75 mg/ml 0.01 %

Ascorbate

F03.1 Na- 150 mg/ml 0.01 %

Ascorbate

F04.1 Thiosulfate 50 mg/ml 0.01 %

F05.1 Thiosulfate 100 mg/ml 0.01 %

The concentration of the protein in the solutions was 1 .78 mg/ml after the addition of the excipient solutions.

The protein solutions were tested for their protein melting point, to assess a potential impact of the additional excipients on this important parameter, since a significant reduction of the protein melting points would compromise protein stability. Protein melting point was determined using Thermofluor (Differential Scanning Fluorimetry) and Micro DSC. The Thermofluor method was performed using a 750 Fast Real Time PCR System (Applied Biosystems). The formulations were added to a fluorescent dye (Sypro Orange) in a 96 well-plate and were analyzed in the PCR System. The temperature was increased from 20°C to 90°C. The melting points of proteins were determined by fluorescence detection. Micro DSC was measured using a VP-DSC (GE Healthcare). The temperature was moved from 20°C to 105°C and the melting point of the protein was determined with the calorimeter. The stability of the solutions was checked by noting the visual appearance and as an additional stability indicating method, the size of the protein was determined using Dynamic Light Scattering (DLS). DLS was performed using a Horiba LB550 (Retsch® Technology). The pH of the solutions was determined using a pH Meter (Mettler Toledo). The hydrodynamic diameter dH (median) as determined by DLS, as well as the visual appearance, the pH and the protein melting points are given are given in table 12. Table 12: Results of stability indicating methods of the protein formulations

^* outlier,

^** method could not be used due to interaction of the fluorescent dye with other additives

No Impact of the excipients in the respective concentrations could be seen with respect to the parameters observed. Neither the protein mp was impacted negatively, nor were the protein diameters changed significantly as a

consequence of the added excipients. DSF could not be used in the presence other additives, since these are interfering with the hydrophobic fluorescent dyes.