ANTIBODY COMPOSITIONS

Abstract

This invention relates to stable formulations that can be prepared to high concentration of active agent using low amounts of detergents and to methods for producing such formulations. In particular, this invention relates to formulations containing the L19-TNFα immunocytokine.

Claims

1. A composition comprising at least 0.4 mg/ml L19-TNFα as set forth in SEQ ID NO: 1, dissolved in a sodium phosphate buffer, wherein the sodium phosphate buffer comprises NaCl at a concentration of at least 20 mM, polysorbate at a concentration of 0.005-0.01% v/v, glycerol at a concentration of 0.5-1.5% w/v, and a stabilizer at a concentration of 10 mM-185 mM, wherein pH of the sodium phosphate buffer is higher than 7.5 and lower than 9.

2. The composition of claim 1, wherein the sodium phosphate buffer comprises NaH.sub.2PO.sub.4 at a concentration of 5-25 mM.

3. The composition of claim 1, wherein the sodium phosphate buffer comprises KCl at a concentration of 1-2 mM.

4. The composition of claim 1, wherein the stabilizer is a sugar.

5. The composition of claim 4, wherein the sugar is selected from the group consisting of mannitol, trehalose, sucrose, sorbitol, maltose and xylitol, or a combination of two or more thereof.

6. The composition of claim 1, wherein NaCl is at a concentration of 20-30 mM.

7. The composition of claim 1, wherein the polysorbate is polysorbate20.

8. The composition of claim 1, wherein the composition is a clear solution without visible particles.

9. The composition of claim 1, wherein the sodium phosphate buffer comprises NaH.sub.2PO.sub.4 at a concentration of 10 mM, mannitol at a concentration of 50 mM, glycerol at a concentration of 1% w/v, KCl at a concentration of 1.5 mM, NaCl at a concentration of 20 mM, and polysorbate20 at a concentration of 0.01% v/v.

10. A composition comprising at least 0.4 mg/ml L19-TNFα as set forth in SEQ ID NO: 1, dissolved in a sodium phosphate buffer, wherein the sodium phosphate buffer comprises a salt at a concentration of at least 1.5 mM, polysorbate at a concentration of 0.005-0.1% v/v, glycerol at a concentration of 0.5-1.5% w/v, and a stabilizer at a concentration of 65 mM-185 mM, wherein pH of the sodium phosphate buffer is higher than 7.5 and lower than 9.

11. The composition of claim 10, wherein the sodium phosphate buffer comprises NaH.sub.2PO.sub.4 at a concentration of 5-25 mM.

12. The composition of claim 10, wherein the sodium phosphate buffer comprises Na.sub.2HPO.sub.4 at a concentration of 5-20 mM.

13. The composition of claim 10, wherein the salt is KCl at a concentration of 1.5-30 mM.

14. The composition of claim 10, wherein the stabilizer is a sugar.

15. The composition of claim 14, wherein the sugar is selected from the group consisting of mannitol, trehalose, sucrose, sorbitol, maltose and xylitol.

16. The composition of claim 10, wherein the sodium phosphate buffer comprises the polysorbate at a concentration of 0.005-0.03%.

17. The composition of claim 10, wherein the polysorbate is polysorbate20.

18. The composition of claim 10, wherein the composition is a clear solution without visible particles.

19. The composition of claim 10, wherein the sodium phosphate buffer comprises NaH.sub.2PO.sub.4 at a concentration of 15 mM, Na.sub.2HPO.sub.4 at a concentration of 10 mM, mannitol at a concentration of 75 mM, glycerol at a concentration of 1% w/v, KCl at a concentration of 1.5 mM, and polysorbate20 at a concentration of 0.01% v/v.

20. The composition of claim 10, wherein the sodium phosphate buffer comprises NaH.sub.2PO.sub.4 at a concentration of 15 mM, Na.sub.2HPO.sub.4 at a concentration of 10 mM, mannitol at a concentration of 75 mM, glycerol at a concentration of 1% w/v, KCl at a concentration of 30 mM, and polysorbate20 at a concentration of 0.01% v/v.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0077] FIG. 1. Schematic structure of the L19-TNFα polypeptide chain (top panel) and amino acid sequence of L19-TNFα (lower panel). (The amino acid sequence of L19-TNFα is set forth in SEQ ID NO:1.)

[0078] FIG. 2. Schematic structure of the L19-IL2 polypeptide chain (top panel) and amino acid sequence of L19-IL2 (lower panel). (The amino acid sequence of L19-IL2 is set forth in SEQ ID NO:2.)

[0079] FIG. 3. Diagram of the cloning scheme for L19-TNFα.

[0080] FIG. 4. Diagram of the cloning scheme for L19-IL2.

[0081] FIG. 5. Absorbance 280 nm of the L19-TNFα immunocytokine in the initial buffer solution at time points in months over a 60 month period. The horizontal line shows the lower detection limit.

[0082] FIG. 6. Western Blot of precipitate arising from the initial L19-TNFα formulation, recovered by centrifugation to yield a pellet that was resuspended and run against dissolved L19-TNFα as a standard. Lane 1: L19-TNFα standard (reducing); lane 2: L19-TNFα standard (non-reducing); lane 3: L19-TNFα pellet (reducing); Lane 4: L19-TNFα pellet (non-reducing).

[0083] FIG. 7. Treatment of a patient with L19-TNFα in phosphate buffer in combination with L19-IL2. CT scan at Day 0 shows the presence of the melanoma lesion (top panel) and CT scan taken six months after the start of treatment, shows that the lesion has completely disappeared (lower panel).

[0084] FIG. 8. Treatment of a patient with L19-TNFα in phosphate buffer in combination with Doxorubicin. A patient with chondrosarcoma was treated with 2 cycles of L19-TNFα (17 ug/kg—3 weekly injections per cycle) and doxorubicin (60 mg/m.sup.2 1 weekly injection per cycle). The target lesion in lung shows growth stabilization at CT after 2 cycles of treatment.

DETAILED DESCRIPTION

[0085] The following applications of the present invention are provided by way of example and not limitation.

[0086] In some embodiments, the composition of the invention may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated.

[0087] Treatments involving the compositions of the invention may include the administration of suitable doses of an anticancer compound. Anti-cancer compounds are cytotoxic compounds which inhibit the growth, division and/or proliferation of cancer cells. Anti-cancer compounds may, in some circumstances, have an effect on normal non-cancer cells in a patient. An anti-cancer compound may, for example, inhibit the cell-cycle or activate apoptosis. Suitable anti-cancer compounds which inhibit the cell cycle include DNA damaging agents and anti-mitotic agents, including inhibitors of mitotic spindle assembly. A DNA damaging agent is a chemotherapeutic compound which induces DNA DSBs in cellular DNA, thereby inhibiting or abolishing DNA replication. Many suitable compounds are known in the art for use in the treatment of cancer, including, for example, bleomycin hydorxyurea, mitomycin and actinomycin and inhibitors of topoisomerase I and II activity, including anthracylines such as daunorubicin, doxorubicin, epirubicin, idarubicin, mitoxantrone and valrubicin, etoposide and teniposide, and members of the tecan family e.g. irinotecan, topotecan, rubitecan. DNA damaging agents may be used as described herein in any convenient form or formulation. For example, any suitable isomer, salt, solvate, chemically protected form, or prodrug of a particular DNA damaging agent may be employed.

[0088] In some preferred embodiments, the DNA damaging agent may be doxorubicin ((8S, 10S)-10-(4-amino-5-hydroxy-6-methyl-tetrahydro-2H-pyran-2-yloxy)-6,8,11-trihydroxy-8-(2-hydroxyacetyl)-1-methoxy-7, 8, 9, 10-tetrahydrotetracene-5,12-dione) such as morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and/or deoxydoxorubicin. Doxorubicin is an anthracycline intercalating agent which is widely used in cancer treatment under trade names such as Adriamycin™, and Rubex™.

[0089] In some embodiments, the composition of the invention may be administered in combination with doxorubicin.

[0090] It is generally convenient to provide the composition of the invention and the doxorubicin as separate molecules. They may be provided as a combined preparation, or as separate formulations to permit either simultaneous or sequential administration. The clinician can determine the most suitable manner of administering the single dose of each L19-TNFα and doxorubicin to the patient.

[0091] For example, the method of treatment may comprise administering the composition of the invention and the doxorubicin in separate injections, simultaneously or sequentially.

[0092] Where sequential administration is used, the immunocytokines are preferably administered within 24 hours, 12 hours, 1 hour or more preferably within 30 minutes of each other.

[0093] The quantity of L19-TNFα administered will depend on the size and nature of the tumour, among other factors. For example, the dose of L19-TNFα may be in the range of 10-17 μg/kg and the dose of doxorubicin 60 mg/m.sup.2. The clinician will determine a therapeutically effective amount for administration.

[0094] Further treatments may be used in combination with the invention include the administration of suitable doses of pain relief drugs such as non-steroidal anti-inflammatory drugs (e.g. aspirin, paracetamol, ibuprofen or ketoprofen) or opiates such as morphine, or antiemetics. Where the immunocytokines are administered for cancer treatment, they are injected parenterally. In one embodiment, the immunocytokines are injected at the site of the tumour, preferably by intratumoural injection. Peritumoural injection, e.g. local intradermal injection, is another suitable method for administering the immunocytokine locally to a tumour site. In some embodiments, the L19-TNFα immunoconjugate may be administered by infusion, e.g. intravenous/systemic infusion. Embodiments of the invention in which the L19-TNFα immunoconjugate is present at low concentration are particularly suited to administration by infusion.

[0095] Treatment of a tumour according to the present invention may include complete eradication of the tumour. The disappearance of any evidence of vital tumour after stopping injections represents complete treatment of the tumour. Disappearance of the tumour may be determined when the tumour has no discernible volume or is no longer visible. Treatment may comprise treatment to eradicate the tumour and prevent tumour regrowth.

[0096] Patients are preferably monitored during a follow-up period of at least one month, preferably at least six months or at least a year, after administration of the immunocytokine combination therapy. Disappearance of the tumour, and lack of tumour regrowth, may be observed in the follow-up period. Absence of tumour regrowth may be observed.

[0097] The quantity of L19-TNFα and the quantity of L19-IL2 administered will depend on the size and nature of the tumour, among other factors. For example, the dose of L19-TNFα may be in the range of 20 μg-2 mg e.g. 100-1,000 μg, 200-600 μg, or between 300-400 μg. The dose may be 50-500 μg (e.g 100-400 μg) per treatment. The dose of L19-IL2 may be in the range of 1-15 Mio IU (e.g. 10-13 Mio IU) per treatment. The treatment may consist of 13 Mio IU of L19-IL2 and 400 μg of L19-TNF in an approximative volume of 2.0 ml as intratumoral injection. The dose of L19-IL2 may be in the range of 20 μg-3 mg e.g. 100-2,500 μg, 300-2,000 μg or between 500-1,800 μg. These are examples only and, of course, different doses may be used. The formulations of this invention enable larger doses to be administered without a large volume being needed. The clinician will determine a therapeutically effective amount for administration.

[0098] The methods may comprise treating a tumour in a patient by injecting L19-TNFα formulation, optionally with the L19-IL2 formulation (administered sequentially or simultaneously) at the tumour site, wherein the tumour disappears in the absence of further doses of the immunocytokine(s). Optionally, further doses may be administered.

[0099] In the event of tumour recurrence after the follow-up period, or if other tumours develop, patients may receive a further treatment with immunocytokine therapy according to the invention, to remove the further tumour.

[0100] Nucleic acid molecules encoding the immunocytokines, and parts thereof, can be used in connection with the compositions and methods of the invention defined in the claims. The nucleic acid molecule may be a vector, e.g. a plasmid suitable for expression of the nucleotide sequence. Normally the nucleotide sequence is operably linked to a regulatory element such as a promoter for transcription. Exemplary vectors for the expression of L19-TNFα and L19-IL2 are disclosed in FIG. 3 and FIG. 4, respectively. The skilled person will appreciate that further DNA vectors capable for expressing the L19-TNFα and L19-IL2 immunocytokines of FIG. 1 and FIG. 2 can be readily constructed. Use of such DNA vectors are also within the scope of this invention when they are used in connection with the compositions and methods defined in the claims.

[0101] The nucleic acid molecules may be contained in a host cell, which may be a cell transfected with the nucleic acid molecules, or a daughter of such a cell. Cells, especially eukaryotic cells e.g. HEK and CHO cells, or bacterial cells e.g. Escherichia coli, containing the nucleic acid molecules can be used.

[0102] Immunocytokines of the invention may be produced using recombinant techniques, for example by expressing recombinant DNA encoding the immunocytokine in a cell. Normally the expression is performed in a eukaryotic host cell containing nucleic acid, as described above. Expression may therefore comprise culturing such a host cell. For L19-TNFα, trimerisation of the subunits may occur in the cell or during purification of the fusion proteins from the cell (21), (24).

Example 1: L19-TNFα Immunocytokine Formulations in Hepes Buffer

[0103] The following formulations were prepared to reach L19-TNFα concentration of 0.4 mg/ml. Formulations were obtained that allows higher concentrations of L19-TNFα to be produced and maintained without precipitation occurring.

[0104] The primary buffer component was Hepes, and the buffer pH, salt and polysorbate concentrations were varied.

[0105] Samples of each L19-TNFα formulation were stored at −80° C.±5° C. and subjected to multiple cycles of freezing and thawing. Three acceptance criteria were used to determine whether each buffer provides an acceptable L19-TNFα formulation:

1. Visual clarity=clear solution without visible particles,
2. A280 stability=less than 5% loss of 280 nm absorbance compared with original value, and
3. Trimer purity=over 95% purity, evaluated by size exclusion chromatography (SEC).

[0106] Hepes buffers were prepared at pH 7.5 and 8.0:

[0107] Hepes-1 comprises 30 mM Hepes at pH 7.5, 5 mM EDTA, 75 mM mannitol and 1.8% glycerol (w/v). None of the acceptance criteria were met.

[0108] Hepes-2 comprises 30 mM Hepes at pH 7.5, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.1% polysorbate20. None of the acceptance criteria were met.

[0109] Hepes-3 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol and 1.8% glycerol (w/v). None of the acceptance criteria were met.

[0110] Hepes-4 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.005% polysorbate20. None of the acceptance criteria were met.

[0111] Hepes-5 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.01% polysorbate20. None of the acceptance criteria were met.

[0112] Hepes-6 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.05% polysorbate20. None of the acceptance criteria were met.

[0113] Hepes-7 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.1% polysorbate20. All three acceptance criteria were met.

[0114] Hepes-8 comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.2% polysorbate20. All three acceptance criteria were met.

[0115] Further formulations were prepared to reach L19-TNFα concentration of 0.2 mg/ml:

[0116] Hepes-A comprises 30 mM Hepes at pH 7.5, 5 mM EDTA, 75 mM mannitol and 1.8% glycerol (w/v). None of the acceptance criteria were met.

[0117] Hepes-B comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol and 1.8% glycerol (w/v). None of the acceptance criteria were met.

[0118] Hepes-C comprises 15 mM Hepes at pH 8.0, 5 mM EDTA, 75 mM mannitol, 1.8% glycerol (w/v) and 0.1% polysorbate20. All three acceptance criteria were met.

[0119] Hepes-based formulations showed particles in suspension, which could only be dissolved in some cases by using 0.1% or 0.2% polysorbate20.

[0120] Following optimization, the most preferred hepes-based buffer for L19-TNFα was 15 mM Hepes, 75 mM Mannitol, 0.2% polysorbate20 (v/v), 1.8% Glycerol (w/v), 5 mM EDTA, pH 8.0—this buffer meets all three acceptance criteria and is denoted Hepes-9.

[0121] L19-TNFα was formulated in the Hepes-9 buffer at a final concentration of 0.092 mg/mL and was tested for stability. This L19-TNFα immunocytokine formulation was stored at −80° for up to 60 months during which time the protein concentration was monitored by measuring the 280 nm absorbance.

[0122] FIG. 5 shows that the 280 nm absorbance does not decrease substantially over the 60 month period, indicating that the initial Hepes-9 buffer is suitable for long-term storage. The horizontal line shows the lower detection limit. The L19-TNFα immunocytokine is stable in the initial buffer stored at −80° for up to 60 months. 1 mg of L19-TNFα corresponds to approximately OD=1.3.

Example 2: Increased Concentration L19-TNFα Formulations

[0123] The Hepes-9 buffer described in Example 1 was not suitable for higher concentrations of L19-TNFα when starting from lower concentrations. For example, when the concentration was increased from 0.092 mg/mL up to 0.4-0.45 mg/mL, small particles appeared in the suspension and a visible pellet was recovered after centrifugation.

[0124] After concentrating the L19-TNFα immunocytokine solution in the Hepes-9 buffer, the product was stored at 2-8° C. and at −80° C. for 24 hours. In both cases small white particles were still visible in suspension. The 280 nm absorbance (A.sub.280nm) of the solution was read after centrifugation and a 6%-8% of OD loss was recorded. Furthermore, when the product was filtered after storage at 2-8° C., a 16% of loss the A.sub.280 nm reading was recorded, as shown in Table 1.

TABLE-US-00001 L19TNFα A.sub.280 nm reading and visual appearance time-point t = 0 0.562 clear solution t = 24 hr 2-8° C. 0.529 (−6%) visible .fwdarw.0.22 μm filtration 0.474 (−16%) clear particles solution t = 24 hr −80° C. 0.519 (−8%) visible particles

[0125] To confirm that the loss of absorbance after concentration was actually due to a precipitation of L19-TNFα, we resuspended the pellet in solution and run a Western blotting, using an anti-TNFα antibody and a chemio-luminescent substrate.

[0126] FIG. 6 shows bands at the molecular weight of L19-TNFα both in the standard solution and in the pellet in both reducing and non-reducing conditions. This indicates that when the concentration of L19-TNFα in the Hepes-9 buffer is increased, the protein precipitates. Lane 1: L19-TNFα standard (reducing); lane 2: L19-TNFα standard (non-reducing); lane 3: L19-TNFα pellet (reducing); Lane 4: L19-TNFα pellet (non-reducing).

Example 4: Various L19-TNFα Immunocytokine Formulations

[0127] The following formulations were prepared to reach L19-TNFα concentration of 0.4 mg/ml. Formulations were obtained that allows higher concentrations of L19-TNFα to be produced and maintained without precipitation occurring.

[0128] The primary buffer component, buffer pH, salt and polysorbate concentrations were varied.

[0129] Samples of each L19-TNFα formulation were stored at −80° C.±5° C. and subjected to multiple cycles of freezing and thawing. Three acceptance criteria were used to determine whether each buffer provides an acceptable L19-TNFα formulation:

1. Visual clarity=clear solution without visible particles,
2. A280 stability=less than 5% loss of 280 nm absorbance compared with original value, and
3. Trimer purity=over 95% purity, evaluated by size exclusion chromatography (SEC).

[0130] Tris-based buffers were prepared at pH 8.0 and 8.5:

[0131] Tris-1 comprises 15 mM Tris at pH 8.0 with 75 mM mannitol and 30 mM NaCl. None of the acceptance criteria were met.

[0132] Tris-2 comprises 15 mM Tris at pH 8.0, 75 mM mannitol, 30 mM NaCl and 1% glycerol (w/v). None of the acceptance criteria were met.

[0133] Tris-3 comprises 15 mM Tris at pH 8.5 and 75 mM mannitol and 30 mM NaCl. None of the acceptance criteria were met.

[0134] Tris-4 comprises 15 mM Tris at pH 8.5, 75 mM mannitol, 30 mM NaCl and 1% glycerol (w/v). None of the acceptance criteria were met.

[0135] Acetate Buffers were Prepared at pH 5:

[0136] Acetate-1 comprises 20 mM NaAc at pH 5.0, 8.5% Sucrose (w/v), 130 PM EDTA. None of the acceptance criteria were met.

[0137] Acetate-2 comprises 20 mM NaAc at pH 5.0, 8.5% Sucrose (w/v), 130 PM EDTA, 0.1% polysorbate20. None of the acceptance criteria were met.

[0138] Histidine buffers were prepared at pH 6, 8 and 9:

[0139] Hist-1 comprises 20 mM histidine at pH 6.0, 8.5% Sucrose (w/v), 130 μM EDTA. None of the acceptance criteria were met.

[0140] Hist-2 comprises 20 mM histidine at pH 8.0, 8.5% Sucrose (w/v), 130 PM EDTA. None of the acceptance criteria were met.

[0141] Hist-3 comprises 20 mM histidine at pH 9.0, 8.5% Sucrose (w/v), 130 PM EDTA. None of the acceptance criteria were met.

[0142] Citrate Buffer was Prepared at pH 6.6

[0143] Citrate-1 comprises 5.6 g/L sodium Citrate, 0.21 g/L citric acid, 70 g/L trehalose dihydrate, 0.2 g/L polysorbate80, 1% (w/v) glycerol, 5 mM EDTA, pH 6.6. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met indicating particles in suspension or aggregation of the trimer.

[0144] Borate Buffer was Prepared at pH 7.4

[0145] Borate-1 comprises 1.1 g/L sodium borate, 3.5 g/L boric acid, 55 mM mannitol, 1% (w/v) glycerol, 5 mM EDTA, pH 7.4. None of the acceptance criteria were met.

[0146] Carbonate Buffer was Prepared at pH 7.0 and pH 8.0

[0147] Carbonate-1 comprises 0.42% (w/v) Na.sub.2CO.sub.3, 5% Glucose, 0.9% (w/v) NaCl, 1% (w/v) glycerol, 1.5 mM KCl, 5 mM EDTA, pH 7.0. None of the acceptance criteria were met.

[0148] Carbonate-2 comprises 0.42% (w/v) Na.sub.2CO.sub.3, 5% Glucose, 0.9% (w/v) NaCl, 1% (w/v) glycerol, 1.5 mM KCl, 5 mM EDTA, pH 8.0. None of the acceptance criteria were met.

[0149] All tris, acetate, histidine, citrate, borate and carbonate-based buffers that were tested showed particles in suspension and were not considered for further investigation.

Example 5: L19-TNFα Immunocytokine Formulations in Phosphate Buffer

[0150] Phosphate buffers were then investigated. Again, samples were prepared to reach L19-TNFα concentration of 0.4 mg/ml. Samples were stored at −80° C.±5° C. and subjected to multiple cycles of freezing and thawing.

[0151] Phosphate buffers were prepared at pH 6.3, 6.5, 7.0 and 8.0 using monobasic sodium phosphate, with and without dibasic sodium phosphate:

[0152] Phos-1 comprises 10 mM NaH.sub.2PO.sub.4 at pH 6.5, 50 mM mannitol, 1% glycerol (w/v) and 1.5 mM KCl. None of the acceptance criteria were met.

[0153] Phos-2 comprises 10 mM NaH.sub.2PO.sub.4 at pH 7.0, 50 mM mannitol, 1% glycerol (w/v) and 1.5 mM KCl. None of the acceptance criteria were met.

[0154] Phos-3 comprises 10 mM NaH.sub.2PO.sub.4 at pH 7.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl and 0.05% polysorbate20. None of the acceptance criteria were met.

[0155] Phos-4 comprises 10 mM NaH.sub.2PO.sub.4 at pH 7.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.05% polysorbate20. Neither the visual clarity nor the A280 stability criteria were met but the trimer purity criteria was met.

[0156] Phos-5 comprises 10 mM NaH.sub.2PO.sub.4 at pH 7.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl and 0.2% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0157] Phos-6 comprises 10 mM NaH.sub.2PO.sub.4 at pH 7.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.2% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0158] Phos-7 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1.5 mM KCl and 30 mM NaCl. None of the acceptance criteria were met.

[0159] Phos-8 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl and 0.05% polysorbate20. None of the acceptance criteria were met.

[0160] Phos-9 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0161] Phos-10 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.02% polysorbate20. Both the trimer purity and A280 stability criteria were met but the visual clarity criteria was not met.

[0162] Phos-11 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.03% polysorbate20. Both the trimer purity and A280 stability criteria were met but the visual clarity criteria was not met.

[0163] Phos-12 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.2% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0164] Phos-13 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 20 mM NaCl and 0.05% polysorbate20. None of the acceptance criteria were met.

[0165] Phos-14 comprises 10 mM NaH.sub.2PO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl and 0.2% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0166] Phos-15 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. Both the visual clarity and trimer purity criteria were met but the A280 stability criteria was not met.

[0167] Phos-16 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 50 mM mannitol, 1.5% glycerol (w/v), 1.5 mM KCl, 10 mM NaCl and 0.01% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0168] Phos-17 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 10 mM NaCl and 0.01% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0169] Phos-18 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 100 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0170] Phos-19 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0171] Phos-20 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 50 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 10 mM NaCl and 0.01% polysorbate20. Both the visual clarity and A280 stability criteria were met but the trimer purity criteria was not met.

[0172] Phos-21 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl, 0.01% polysorbate20 and 5 mM EDTA. All three acceptance criteria were met.

[0173] Phos-22 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM saccharose, 1.5 mM KCl, 30 mM NaCl, 1% (w/v) glycerol, 0.01% (v/v) polysorbate20 and 5 mM EDTA. All three acceptance criteria were met.

[0174] Phos-23 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 70 g/L trehalose dihydrate (i.e. 185 mM trehalose), 1.5 mM KCl, 30 mM NaCl, 1% (w/v) glycerol, 0.01% (v/v) polysorbate20 and 5 mM EDTA. All three acceptance criteria were met.

[0175] Phos-24 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1.5 mM KCl, 30 mM NaCl, 1% (w/v) glycerol, 0.01% polysorbate80 and 5 mM EDTA. All three acceptance criteria were met.

[0176] Phos-25 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1.5 mM KCl, 30 mM NaCl, 1% (w/v) glycerol, 0.02% (w/v) polyoxyl 35 castor oil and 5 mM EDTA. None of the acceptance criteria were met.

[0177] Phos-26 comprises 6.7 mM Na.sub.2HPO.sub.4 at pH 6.3, 133 mM mannitol, 1.8 mM KCl, 20 mM NaCl and EDTA 5 mM. Phos-26 is a comparative example, based on a prior art buffer that was denoted “PBS-Siena” in WO2007/128563 (27). None of the acceptance criteria were met.

[0178] Phos-27 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.03% polysorbate20. All of the acceptance criteria were met.

[0179] Most preferably all three of the acceptance criteria are met.

[0180] The tests using e.g. Phos-22 and Phos-23 show that, regardless of the type of stabilizer, high quality soluble L19-TNFα solutions can be maintained.

[0181] Reduction of L19-TNFα concentration in each buffer has not been observed to reduce formulation stability.

[0182] The most preferred buffers selected for ongoing clinical tests were Phos-19 and Phos-21. The difference between these buffers is the presence of EDTA in Phos-21 and the absence of EDTA in Phos-19. While the presence or absence of EDTA is not observed to impact the solubility of L19-TNFα, nor the stability of the L19-TNFα solution, EDTA may be helpful as a preservative.

[0183] The importance of KCL was further investigated. Phos-19 Phosphate buffer was prepared without KCl as follows.

[0184] Phos-28 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0185] It was found that KCl was not essential in the formulation. A KCl concentration in the range about 1.5-1.8 mM helped to maintain the isotonicity of the solution by preventing a sudden pH shift during freezing of the formulation.

[0186] It was found that in absence of polysorbate the samples were not stable (e.g. Phos-26, borate-1 or Phos-25). (In Phos-25 polysorbate is replaced with the surfactant, polyoxyl 35 castor oil.)

Example 6: Effect of the pH on Phos-19 Phosphate Buffer

[0187] Phosphate buffer Phos-19 was further investigated regarding the pH.

[0188] Phosphate buffer Phos-19 was prepared at pH 6.5, 7.0, 7.5, 7.8, 8.5, 8.8 and 9.0.

[0189] Phos-29 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 6.5, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. None of the acceptance criteria were met.

[0190] Phos-30 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 7.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. None of the acceptance criteria were met.

[0191] Phos-31 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 7.5, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. None of the acceptance criteria were met.

[0192] Phos-32 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 7.8, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0193] Phos-33 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.5, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0194] Phos-34 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.8, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met

[0195] Phos-35 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 9.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. None of the acceptance criteria were met.

[0196] It was therefore found that only pH values above 7.5 and below 9 are suitable for phosphate buffer formulations

Example 7: Studies on the Salt and the Phosphate Concentration in the Phos-19 Phosphate Buffer

[0197] The salt NaCl was substituted by another salt (e.g. KCl). Nacl was removed from Phos-19 and substituted by KCl at a concentration of 1.5 mM or 30 mM.

[0198] Phos-36 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0199] Phos-37 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 30 mM KCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0200] It was therefore demonstrated that different salts can be used in phosphate buffer formulations.

[0201] Phosphate buffer (Phos-19 variants) comprising various concentrations of NaH.sub.2PO.sub.4 and Na.sub.2HPO.sub.4 were tested.

[0202] Phos-38 comprises 10 mM NaH2PO4 and 10 mM Na2HPO4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0203] Phos-39 comprises 20 mM NaH2PO4 and 10 mM Na2HPO4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0204] Phos-40 comprises 15 mM NaH2PO4 and 15 mM Na2HPO4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 1.5 mM KCl, 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

Example 8: Further Studies on Phosphate Buffers, Regarding Glycerol, KCl, and Tween80

[0205] Further investigations using the Phos-28 (15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 30 mM NaCl and 0.01% polysorbate20) were done. Phos-28 corresponds to Phos-19 without KCl.

(i) Various concentrations of glycerol (w/v) were tested:
Glycerol concentrations of 0.5% and 1.5% (w/v) were tested.

[0206] Phos-41 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 0.5% glycerol (w/v), 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0207] Phos-42 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1.5% glycerol (w/v), 30 mM NaCl and 0.01% polysorbate20. All of the acceptance criteria were met.

[0208] It was therefore found that a concentration of glycerol as low as 0.5% can be used in phosphate buffer formulations that pass all three acceptance criteria.

(ii) Various Concentrations of Tween80:

[0209] Phosphate buffer (Phos-28) comprising various concentrations of Tween80 instead of Tween20 were tested.

[0210] Phos-43 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 30 mM NaCl and 0.005% polysorbate80. All of the acceptance criteria were met.

[0211] Phos-44 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 30 mM NaCl and 0.03% polysorbate80. All of the acceptance criteria were met.

[0212] Phos-45 comprises 15 mM NaH.sub.2PO.sub.4 and 10 mM Na.sub.2HPO.sub.4 at pH 8.0, 75 mM mannitol, 1% glycerol (w/v), 30 mM NaCl and 0.1% polysorbate80. All of the acceptance criteria were met.

[0213] It was therefore found that a concentration of polysorbate 80 as low as 0.005% can be used for phosphate buffer formulations.

Example 9: Combining L19-TNFα and L19-IL2 Formulations

[0214] It was then investigated if the new formulation buffer of L19-TNFα dissolved in Phos-19 was suitable for combination with L19-IL2 in the clinical setting.

[0215] Various mixes of L19-TNFα in the Phos-19 buffer were performed with L19-IL2 in a buffer comprising 6.7 mM NaH.sub.2PO.sub.4, 20 mM NaCl, 1.8 mM KCl, 133 mM mannitol, 0.1% polysorbate80 (v/v), 1% glycerol (w/v) and 5 mM EDTA and has pH 6.3. These formulations were mixed together and the resultant mixtures were tested both at time 0 and after 3 hours at room temperature. We considered that in the clinical operations, the mixture would in no case exceed 3 hours of incubation as the two products are mixed and injected almost immediately. The mixtures were prepared by adding L19-TNFα into the L19-IL2 solution and vice versa. The results are shown in Table 2.

TABLE-US-00002 TABLE 2 Calculated Osmolality TNFα IL2 Time point Sample Protein Amount § mOsmol/Kg pH % IR Bioactivity * Bioactivity * SDS PAGE t = 0 Mix 1 L19TNFa: 0.46 mg 336 7.24 >99%  95% 94% L19TNFa and 0.4 mg L19TNFa + L19IL2: 2.18 mg L19IL2 band 2.17 mg L19IL2 visible Mix 2 L19IL2: 2.11 mg 329 7.36 >99% 116% 90% L19TNFa and 2.17 mg L19IL2 + L19TNFa: 0.45 mg L19IL2 band 0.4 mg L19TNFa visible t = 3 hr RT Mix 1 L19TNFa: 0.42 mg n/a n/a >99% 109% 89% L19TNFa and 0.4 mg L19TNFa + L19IL2: 2.07 mg L19IL2 band 2.17 mg L19IL2 visible Mix 2 L19IL2: 2.18 mg n/a n/a >99% 112% 86% L19TNFa and 2.17 mg L19IL2 + L19TNFa: 0.45 mg L19IL2 band 0.4 mg L19TNFa visible

[0216] Protein amount was calculated by quantitative Cation Exchange chromatography. Bioactivity was calculated by referring to TNFα or IL2 standard. IR=Immunoreactivity

[0217] Importantly, a clear solution with no protein loss or aggregates was recovered after the 3 hours. Overall the mixture was found to be stable.

Example 10: Treatment of a Patient with L19-TNFα in Phosphate Buffer in Combination with L19-IL2

[0218] The new formulation of L19-TNFα dissolved in Phos-21 in combination with L19-IL2, which is in a buffer comprising 6.7 mM NaH.sub.2PO.sub.4, 20 mM NaCl, 1.8 mM KCl, 133 mM mannitol, 0.28% polysorbate80 (v/v), and 1% glycerol (w/v) and which has pH 6.3, was then tested in a clinical setting.

[0219] A 59 year old patient was diagnosed with Stage IIIB melanoma and presented an injectable retroauricolar metastasis.

[0220] The patient received four treatments through intralesional injection with L19-TNFα dissolved in Phos-21 in combination with L19-IL2. First and second treatments consisted of a dose of 13 Mio IU of L19IL2 and 400 μg of L19-TNFα. The third was ¾ of the dose of L19-TNFα and L19-IL2 and the fourth treatment was ½ of the dose of L19-TNFα and L19-IL2.

[0221] Results

[0222] The CT scan at Day 0 shows the presence of the melanoma lesion (FIG. 5, top panel). The CT scan taken six months after the start of treatment, shows that the lesion has completely disappeared (FIG. 5, lower panel).

Example 11: Treatment of a Patient Having a Sarcoma with L19-TNFα in Phosphate Buffer in Combination with Doxorubicin

[0223] A patient with chondrosarcoma was treated with 2 cycles of L19-TNFα (17 ug/kg—3 weekly injections per cycle) and doxorubicin (60 mg/m.sup.2 1 weekly injection per cycle), administered by infusion.

[0224] The target lesion in lung showed growth stabilization at CT after 2 cycles of treatment (FIG. 8).

[0225] Sequences

TABLE-US-00003 SEQ ID NO: 1 = L19-TNFQ EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSS ISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPF PYFDYWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLS CRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSSSGSSSSGSS SSGVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQ LVVPSEGLYLIYSQVLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAI KSPCQRETPEGAEAKPWYEPIYLGGVFQLEKGDRLSAEINRPDYLDFAES GQVYFGIIAL SEQ ID NO: 2 = L19-IL2 EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSS ISGSSGTTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPF PYFDYWGQGTLVTVSSGDGSSGGSGGASEIVLTQSPGTLSLSPGERATLS CRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRATGIPDRFSGSGSGTDF TLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSSSGSSSSGSS SSGAPTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMP KKATELKHLQCLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT

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