This application claims priority from European Patent Application No. 22163383.7 filed on 21 March 2022, the contents and elements of which are herein incorporated by reference for all purposes.

Field of the Invention

The present invention relates to a preparation, in particular a pharmaceutical preparation, comprising a non-glycosylated TNFa immunoconjugate and a glycosylated TNFa immunoconjugate, wherein the percentage of the glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the preparation is low, preferably 10% or lower. The preparation finds application in the treatment of cancer, such as brain tumors, in human patients.

Background

Current applicants described in W02001/062298, which is hereby incorporated by reference in its entirety, an immunoconjugate comprising human TNFa fused to antibody L19 (“L19-TNFa”). L19 (US patent n° 8,097,254) specifically binds the ED-B domain of fibronectin isoform B-FN, which is one of the best-known markers of angiogenesis. ED-B is an extra domain of 91 amino acids found in the B-FN isoform. ED-B accumulates around neovascular structures in aggressive tumours and other tissues undergoing angiogenesis, such as the endometrium in the proliferative phase and some ocular structures in pathological conditions but is otherwise undetectable in normal adult tissues. A number of uses, including the treatment of human cancers, and formulations of L19-TNFa have been previously described by current applicants in WO2013/045125, WO2018/011404, WO2019/185792, WO2021/234178, which are hereby incorporated by reference in their entirety.

Large-scale production of immunoconjugates, such as L19-TNFa can be achieved by recombinant expression. However, prokaryotic (bacterial) expression is unsuitable for the expression of complex proteins such as L19-TNFa which forms a large 150 kDa non-covalent homotrimer in solution.

Borsi et al. Blood (2003) 102, 4384 detail the use SP2/0 murine myeloma cells to express recombinant L19-murine(m)TNFa, reporting a final yield of 3mg/L of purified protein which is not suitable for industrial production of the conjugate.

In light of the above, there is a need in the art for additional methods for large-scale production of L19-human TNFa suitable for the treatment of human patients. Summary of the Invention

The present inventors have devised a production method for the preparation of L19-TNFa, which has a high yield and, surprisingly, results in a low percentage of glycosylated L19-TNFa in the preparation. Specifically, the percentage of glycosylated L19-TNFa in the preparation was reported to be between 3% and 5.36% (Figure 5A)., with an average of 4.19% of the L19-TNFa being glycosylated (Figure 5B).

Furthermore, preparations comprising a low percentage of glycosylated L19-TNFa are expected to have superior biological activity in comparison with preparations comprising a high percentage of glycosylated L19-TNFa. Specifically, L19-TNFa conjugates which are not glycosylated are expected to have increased affinity for Tumor Necrosis Factor Receptor 1 (TNFR1) and or Tumor Necrosis Factor Receptor 2 (TNFR2) relative to glycosylated L19-TNFa conjugates.

Preparations comprising a low percentage of glycosylated L19-TNFa are also expected to have superior thermostability and/or storage stability in comparison with preparations comprising a high percentage of glycosylated L19-TNFa. Specifically, L19-TNFa conjugates which are not glycosylated are expected to have increased thermostability relative to glycosylated L19-TNFa conjugates.

Still further, preparations comprising a low percentage of glycosylated L19-TNFa are expected to be less susceptible to protease-mediated degradation in comparison with preparations comprising a high percentage of glycosylated L19-TNFa. Specifically, L19-TNFa conjugates which are not glycosylated are expected to be less susceptible to protease-mediated degradation relative to glycosylated L19-TNFa conjugates.

Preparations comprising a low percentage of glycosylated L19-TNFa are also expected to be more suitable for lyophilisation in comparison with preparations comprising a high percentage of glycosylated L19-TNFa.

Without wishing to be bound by theory, it is believed that the fermentation conditions used to prepare the L19-TNFa conjugate described herein result in the low level of glycosylated species observed.

High levels of glycosylation present challenges in terms of batch-to-batch reproducibility for good manufacturing practice (GMP) production, increased immunogenicity and suboptimal pharmacokinetics when preparing a product for industrial development. Durocher & Butler, Current Opinion Biotechnol (2009), 20, 770 explain that glycan structures may be immunogenic and that immunogenicity can reduce the efficacy of a biologic through rapid clearance by immune system. Planinc et al. Analytica Chimica Acta (2016) 13, 27 similar confirm the importance of low abundance of glycans due to their potential immunogenicity. Leepenies & Seeberger Nature Biotechnol. (2016), 32, 443 further comment that heterogeneous glycosylation can result in batch-to-batch variations in efficacy or pharmacokinetics.

In view of the drawbacks associated with glycosylation of immunoconjugates such as L19-TNFa detailed above, the low percentage of glycosylated L19-TNFa in the preparation resulting from the production method devised by the present inventors is expected to have advantageous properties when used in the treatment of human patients compared with L19-TNFa preparations comprising higher percentages of glycosylated L19-TNFa.

The low level of glycosylated L19-TNFa in the preparation was surprising in view of the fact that production of human TNFa when prepared in the same cell line (Chinese Hamster Ovary) had previously been shown to result in high levels of glycosylation. Specifically, in WO98/55662, expression of recombinant non-conjugated human TNFa in Chinese Hamster Ovary (CHO) cells resulted in a TNFa preparation in which 35% of the total TNFa was reported to be O- glycosylated. The authors consider this to be advantageous on the basis that glycosylated TNFa may have an increased half-life in body fluids, may improve binding to receptors and may be better protected against the influence of proteases than non-glycosylated TNFa. In Takakura et al., Eur J Bioch (1996), 235, 431 , the human B-cell lymphoblastoid cell line BALL-1 was used to express recombinant human TNFa. Consistent with the high level of glycosylation reported in WO98/55662, the percentage of O-glycosylated TNFa in the resulting preparation was reported to be 20%. The O-glycosylation was further reported to be linked to the serine at position 4 of the amino acid sequence of human TNFa.

Thus, in a first aspect, the present invention provides a preparation comprising:

(i) a non-glycosylated TNFa immunoconjugate; and

(ii) a glycosylated TNFa immunoconjugate; wherein the TNFa immunoconjugate comprises human TNFa conjugated to an antibody molecule comprising the L19 complementarity determining regions (CDRs) set forth in SEQ ID NOs 1 to 6, and wherein the percentage of the glycosylated TNFa immunoconjugate is < 10% of the total TNFa immunoconjugate in the preparation. In a preferred embodiment, the antibody molecule in the TNFa immunoconjugate comprises the L19 VH domain set forth in SEQ ID NO: 7 and the L19 VL domain set forth in SEQ ID NO: 9. More preferably, the antibody molecule has the sequence of the L19 antibody in scFv format set forth in SEQ ID NO: 10. Most preferably, the TNFa immunoconjugate consists of or comprises the sequence of L19-TNFa set forth in SEQ ID NO: 13.

The TNFa is preferably human TNFa. Most preferably, the TNFa has the sequence set forth in SEQ ID NO: 11.

Thus, in a preferred embodiment, the present invention provides a preparation comprising:

(i) non-glycosylated L19-TNFa; and

(ii) glycosylated L19-TNFa; wherein the L19-TNFa comprises or consists of the sequence set forth in SEQ ID NO: 13; and wherein the percentage of glycosylated L19-TNFa is < 10% of the total L19-TNFa in the preparation.

The percentage of glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the preparation is preferably < 10%, < 9%, < 8%, < 7%, or < 6%, with a lower % of glycosylation being preferred for the reasons explained above.

The percentage of glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the preparation may be > 2%, or > 3%.

For example, the percentage of glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the preparation may be > 3% and < 6%.

The glycosylated TNFa immunoconjugate preferably comprises an O-linked glycosylation at the serine at position 4 of TNFa, wherein the TNFa has the sequence set forth in SEQ ID NO: 11. Where the TNFa immunoconjugate is L19-TNFa and comprises or consists of the sequence set forth in SEQ ID NO: 13, the glycosylated L19-TNFa preferably comprises an O-linked glycosylation on the serine at position 257 of SEQ ID NO: 13. The O-linked glycosylation preferably comprises one core of hexose-acetyl-hexose 1 and one N-acetylneuraminic acid residue. Most preferably, the O-linked glycosylation is HexNAciHexiNeuAci. In a preferred embodiment, the TNFa immunoconjugate is not glycosylated at any other position in the TNFa sequence. In a preferred embodiment, the present invention provides a preparation comprising:

(i) non-glycosylated L19-TNFa; and

(ii) glycosylated L19-TNFa; wherein the L19-TNFa comprises or consists of the sequence set forth in SEQ ID NO: 13, wherein the percentage of glycosylated L19-TNFa is < 6% of the total L19-TNFa in the preparation, and wherein the preparation has a higher biological activity compared to a preparation in which the percentage of glycosylated L19-TNFa is 15% or more of the total L19-TNFa in the preparation.

In a preferred embodiment, the preparation has increased affinity for Tumor Necrosis Factor Receptor 1 (TNFR1) and or Tumor Necrosis Factor Receptor 2 (TNFR2) relative to a preparation in which the percentage of glycosylated L19-TNFa is 15% or more of the total L19- TNFa in the preparation.

In a preferred embodiment, the preparation has superior thermostability and/or storage stability compared to a preparation in which the percentage of glycosylated L19-TNFa is 15% or more of the total L19-TNFa in the preparation.

In a preferred embodiment, the preparation is less susceptible to protease-mediated degradation compared to a preparation in which the percentage of glycosylated L19-TNFa is 15% or more of the total L19-TNFa in the preparation.

In a preferred embodiment, the preparation is more suitable for lyophilisation compared to a preparation in which the percentage of glycosylated L19-TNFa is 15% or more of the total L19- TNFa in the preparation.

Methods for determining the type and percentage of glycosylated TNFa immunoconjugate in a preparation of the TNFa immunoconjugate are known to the skilled person and include mass spectrometry, in particular intact mass analysis, as described herein.

The pharmaceutical preparation of the invention may further comprise, in addition to the active ingredient (glycosylated and non-glycosylated TNFa immunoconjugate), a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art and suitable for the administration to human patients. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. For injection at a tumour site, the pharmaceutical preparation may be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. The pharmaceutical preparation of the invention may be for use in a method of treating cancer in a patient. Also provided is a method of treating cancer, the method comprising administering a therapeutically effective amount of the pharmaceutical preparation of the invention to the patient. Similarly provided is the use of the pharmaceutical preparation of the invention in the manufacture of a medicament for the treatment of cancer. The patient is preferably a human patient.

Cancers that may be treated using a pharmaceutical preparation of the invention include skin cancer, such as a malignant melanoma or non-melanoma skin cancer, sarcoma, such as soft- tissue sarcoma, and brain tumors such as glioma or glioblastoma.

The invention includes the combination of the aspects and preferred features described except where such a combination is clearly impermissible or expressly avoided.

Summary of the Figures

Embodiments and experiments illustrating the principles of the invention will now be discussed with reference to the accompanying figures in which:

Figure 1 shows the general outline of the production process of L19-TNFa used to obtain the pharmaceutical preparation comprising a low percentage of glycosylated L19-TNFa of the invention.

Figure 2 shows the general fermentation procedure of the production process for L19-TNFa used to obtain the pharmaceutical preparation comprising a low percentage of glycosylated L19-TNFa of the invention.

Figure 3 A and B: bidimensional SDS-PAGE analysis of the pharmaceutical preparation comprising a low percentage of glycosylated L19-TNFa confirmed the purity of the sample after production, as well as the molecular weight (~45 KDa) and isoelectric point (~7.7) of L19-TNFa. Two spots were visible: non-glycosylated L19-TNFa (spot 1) and glycosylated L19-TNFa (spot 2). Non-glycosylated L19-TNFa was the most intense spot, while the spot corresponding to glycosylated L19-TNFa was barely visible, indicating a very low abundance of the glycosylated species. C: purified milligrams of L19-TNFa per litre of fermentation culture recovered from seven lots of production. Figure 4 shows the deconvoluted spectrum of the mass spectrometry analysis of a L19-TNFa preparation comprising a low percentage of glycosylated L19-TNFa prepared according to the method described herein. The main peak (1) at 43957 Da corresponds to the non-glycosylated L19-TNFa conjugate while peak (2) at 44613 Da corresponds to the L19-TNFa variant with an O-linked glycosylation at position 257. The other minor peaks are irrelevant: the small peak at 43913 Da is a typical MS artefact corresponding to L19-TNFa without one CO2 group. The other small peak at 44056 Da corresponds to L19-TNFa in aggregation with a phosphate group of the buffer.

Figure 5A shows the percentage of non-glycosylated L19-TNFa and glycosylated L19-TNFa of the total L19-TNFa in different production lots of the pharmaceutical preparation prepared using the method described herein. In seven lots of production using the method described herein, glycosylated L19-TNFa never exceeded 6% of the total L19-TNFa in the preparation, with an average percentage of glycosylated L19-TNFa in the preparation of 4.19% (Figure 5B).

Figure 6 shows the MS1 spectrum of the glycosylated TNFa peptide SSSRTPSDKPVAHWANPQAEGQLQWLNR in a representative lot of production of an L19- TNFa preparation comprising a low percentage of glycosylated L19-TNFa. Detection of a 4+ charged ion at 958.21 m/z is consistent with the presence of one additional NeuAc group on the HexNAcHexI core attached to the serine at position 2 of the TNFa peptide which corresponds to the serine at position 257 in the L19-TNFa conjugate.

Figure 7 shows the MS2 spectrum of the glycosylated TNFa peptide SSSRTPSDKPVAHWANPQAEGQLQWLNR in a representative lot of production of an L19- TNFa preparation comprising a low percentage of glycosylated L19-TNFa. The precursor ion at 958.21 m/z has been isolated and HCD fragmented to generate this MS2 spectrum. Together with the expected list of y-ions, the presence of two peaks at 274.09 m/z (NeuAc-H2O) and 292.10 m/z (NeuAc) confirms the presence of the NeuAc group on the peptide. This data further confirms the correct identification of the HexNAciHexiNeuAci group on the serine at position 2 of the peptide, which corresponds to the serine at position 257 of L19-TNFa conjugate.

Detailed Description

Aspects and embodiments of the present invention will now be discussed with reference to the accompanying figures. Further aspects and embodiments will be apparent to those skilled in the art. All documents mentioned in this text are incorporated herein by reference. The pharmaceutical composition of the present invention comprises a TNFa immunoconjugate, wherein the TNFa immunoconjugate comprises an antibody molecule comprising the L19 complementarity determining regions (CDRs) set forth in SEQ ID NOs 1 to 6 and TNFa.

The antibody molecule is preferably monoclonal. The antibody molecule may be human or humanised, but preferably is a human antibody molecule.

The TNFa immunoconjugate may be isolated, in the sense of being free from contaminants, such as antibodies able to bind other polypeptides, and/or serum components.

The antibody molecule may be natural or partly or wholly synthetically produced. For example, the antibody molecule may be a recombinant antibody molecule.

The antibody molecule may be an immunoglobulin, or an antigen-binding fragment thereof. For example, the antibody molecule may be an IgG, IgA, IgE or IgM molecule, preferably an IgG molecule, such as an lgG1, lgG2, lgG3 or lgG4 molecule, but more preferably is an antigenbinding fragment thereof. In a more preferred embodiment, the antibody molecule comprises or consists of a single-chain Fv (scFv), a small immunoprotein, a diabody, but most preferably is an scFv.

The antibody molecule preferably comprises the L19 VH domain set forth in SEQ ID NO: 7 and/or the L19 VL domain set forth in SEQ ID NO: 9. More preferably, the antibody molecule has the sequence of the L19 antibody in scFv format set forth in SEQ ID NO: 10.

Where the antibody molecule is an scFv, the VH and VL domains of the antibody are preferably linked by a 12 to 20 amino acid linker. For example, the VH and VL domains may be linked by an amino acid linker which is 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acid in length. Suitable linker sequences are known in the art and include the linker sequence set forth in SEQ ID NO: 8.

The TNFa is preferably human TNFa. Most preferably, the TNFa comprises or consists of the sequence set forth in SEQ ID NO: 11.

The antibody molecule, e.g. in scFv format, and the TNFa may be connected to each other directly, for example through any suitable chemical bond, but preferably are connected via a peptide linker. The chemical bond may be, for example, a covalent or ionic bond. Examples of covalent bonds include peptide bonds (amide bonds) and disulphide bonds. Where the TNFa is connected to the antibody molecule via a peptide linker, the peptide linker may be a short (2-30, preferably 10-20) residue stretch of amino acids. Suitable examples of peptide linker sequences are known in the art. One or more different linkers may be used. An exemplary linker sequence is set forth in SEQ ID NO 12. In one embodiment, the linker may be a cleavable linker.

Where the antibody molecule and TNFa are connected via a peptide bond or peptide linker, the conjugate may be produced (secreted) as a single chain polypeptide, such as a fusion protein.

In a preferred embodiment, the TNFa is conjugated to the C-terminus of the antibody molecule in scFv format. Most preferably, the TNFa immunoconjugate consists of, or comprises, the sequence of L19-TNFa set forth in SEQ ID NO: 13.

The TNFa in the glycosylated TNFa immunoconjugate comprised in the pharmaceutical preparation of the invention, preferably comprises an O-linked glycosylation on the serine at position 4 of the TNFa sequence set forth in SEQ ID NO: 11 . Where the TNFa immunoconjugate is L19-TNFa and comprises or consists of the sequence set forth in SEQ ID NO: 13, the glycosylated L19-TNFa preferably comprises an O-linked glycosylation on the serine at position 257 of SEQ ID NO: 13.

The O-linked glycosylation comprises one core of hexose-acetyl-hexose 1 and one N- acetylneuraminic acid residue. Preferably, the O-linked glycosylation is HexNAciHexiNeuAci. In a preferred embodiment, the TNFa immunoconjugate is not glycosylated at any other position in the TNFa sequence.

The L19 in the glycosylated L19-TNFa immunoconjugate comprised in the pharmaceutical preparation of the invention is not glycosylated. That is, only the TNFa in the glycosylated L19- TNFa immunoconjugate comprised in the pharmaceutical preparation of the invention is glycosylated.

The percentage of glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the pharmaceutical preparation is preferably < 10%, < 9.5%, < 9%, < 8.5%, < 8%, < 7.5%, < 7%, < 6.5%, < 6%, or < 5.5%, with a lower % of glycosylation being preferred for the reasons explained above. In a preferred embodiment, the % of glycosylation is < 6%, more preferably < 5.5%. The percentage of glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the preparation may be > 2%, > 2.5%, or > 3%. This excludes TNFa immunoconjugate made in bacterial cells, which are not glycosylated.

For example, the percentage of glycosylated TNFa immunoconjugate of the total TNFa immunoconjugate in the preparation may be > 2% and < 10%, > 2% and < 6%, > 3% and < 6%, > 2% and < 5.5%, > 3% and < 5.5%.

Methods for measuring the percentage of glycosylated TNFa immunoconjugate present in a pharmaceutical composition, as well as determining the type of glycosylation present are known in the art and include mass spectrometry, in particular intact mass analysis. An exemplary method is detailed in Example 3. The results of said analysis are shown in Figures 4 to 7.

The pharmaceutical preparation of the invention may comprise, in addition to glycosylated and non-glycosylated TNFa immunoconjugate, as set out in the claims, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art and suitable for the administration to human patients. The pharmaceutical composition may comprise a buffer composition as disclosed in W02018/011404. In particular, the pharmaceutical composition may comprise glycosylated and non-glycosylated TNFa immunoconjugate, as set out in the claims, dissolved in a sodium phosphate buffer which comprises a salt at a concentration of at least about 1.5 mM, polysorbate at a concentration of at least about 0.005% (v/v), and a stabilizer, wherein the pH of the sodium phosphate buffer is higher than 7.5 and lower than 9. Preferably, the polysorbate concentration is about 0.1% (v/v) or less. The sodium phosphate buffer preferably comprises glycerol at about 0.5-1.5% w/v. The sodium phosphate buffer may further comprises NaH2PO4 at a concentration of about 5-25 mM. The sodium phosphate buffer may further comprises Na2HPO4 at a concentration of about 5-20 mM. The sodium phosphate buffer may also comprise KCI at a concentration of about 1-2 mM. Additionally, the sodium phosphate buffer may comprise EDTA at a concentration of about 1-20 mM. The stabilizer may be a sugar, preferably mannitol, trehalose, sucrose, sorbitol, maltose or xylitol, most preferably mannitol. The sugar may be present at a concentration of 20- 250 mM. The salt may be NaCI and may be present in the sodium phosphate buffer at a concentration of about 10-30 mM. The sodium phosphate buffer preferably comprises polysorbate at a concentration of about 0.005 to about 0.03%. The polysorbate may be polysorbate20. In a preferred embodiment, the concentration of the total TNFa immunoconjugate (glycosylated and non-glycosylated) in the pharmaceutical preparation may be is at least about 0.2 mg/mL, more preferably at least about 0.4 mg/mL. In a most preferred embodiment, the pharmaceutical preparation of the invention comprises, in addition to glycosylated and non-glycosylated TNFa immunoconjugate, NaH2PO4(2H2O) at a concentration of 15mM, Na2HPC>4(2H2O) at a concentration of 10mM, KCI at a concentration of 1.5mM, mannitol at a concentration of 75mM, NaCI at a concentration of 30mM, 1 % w/v glycerol, EDTA at a concentration of 5mM, 0.01% v/v Tween20, and has a pH of 8.00.

The preparation of the invention is expected to have a higher biological activity compared to a preparation in which the percentage of glycosylated L19-TNFa is 15% or more, 20% or more, 25% or more, or 30% or more, of the total L19-TNFa in the preparation.

In one embodiment, the preparation is expected to have a higher affinity for TNFR1 and/or TNFR2 as measured by BiaCore or other suitable techniques.

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

Treatments involving the pharmaceutical preparation of the invention may include the administration of a second anti-cancer therapy. Many suitable anti-cancer therapies are known in the art. For example, L19-TNFa has been successfully used in anti-cancer therapy when administered in combination with doxorubicin, dacarbazine, radiotherapy and temozolomide, or lomustine (WO2021/234178). Clinical trials for these combinations are ongoing. Promising anticancer effects have also been reported for the combined administration of L19-TNFa and L19- IL2 (W02018/011404). This is in addition to the anti-cancer effect demonstrated for L19-TNFa monotherapy.

Thus, the pharmaceutical preparation of the invention may be for use in a method of treating cancer in a patient, wherein the method further comprises administering a second anti-cancer therapeutic selected from the group consisting of doxorubicin, dacarbazine, radiotherapy and temozolomide, lomustine, or L19-IL2, to the patient. Administration of the pharmaceutical preparation of the invention and the second anti-cancer therapy to the patient may be simultaneous or sequential, whereby simultaneous administration refers to administration in the same treatment cycle but not necessarily on the same day.

The pharmaceutical preparation of the invention and the second anti-cancer therapy may be provided as a combined preparation but are preferably provided as separate preparations to permit either simultaneous or sequential administration. Where the treatment further involves radiotherapy, this will necessarily be administered separately from the pharmaceutical preparation of the invention but administration may nonetheless be simultaneous.

Further treatments may be used in combination with the pharmaceutical preparation of 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 pharmaceutical preparation of the invention is administered for cancer treatment, it may be injected parenterally. In one embodiment, the pharmaceutical preparation of the invention is injected at the site of the tumour, preferably by intratumoral injection. Peritumoral injection, e.g. local intradermal injection, is another suitable method for administering the pharmaceutical preparation of the invention locally to a tumour site. In some embodiments, the pharmaceutical preparation of the invention may be administered by infusion, e.g. intravenous infusion.

Cancer treatment according to the present invention may include complete eradication of the tumour. The disappearance of any evidence of vital tumour after termination of the treatment 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.

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 pharmaceutical preparation of the invention. Disappearance of the tumour, and lack of tumour regrowth, may be observed in the follow-up period. Absence of tumour regrowth may be observed.

The quantity of L19-TNFa administered through administration of the pharmaceutical preparation of the invention to the patient will depend on the size and nature of the tumour, among other factors. The determination of suitable doses is within the competence of the skilled practitioner. Suitable doses are disclosed in WO2021/234178, W02018/011404, and WO2013/045125. In particular, L19-TNFa can be given at doses between 5 and 20 pg/Kg, preferably between 6 and 18 pg/Kg, between 7 and 17 pg/Kg or between 8 and 15 pg/Kg, more preferably between 10 and 13 pg/Kg. Preferably, the dose of L19-TNFa is in the range of 200 pg to 400 pg. Most preferably, the dose of L19-TNFa is 400 pg. These are examples only and different doses may be used. The pharmaceutical preparation of the invention may be for use in a method of treating cancer. The cancer may be a solid tumor. In a preferred embodiment, the cancer is selected from a skin cancer such as malignant melanoma, non-melanoma skin cancer such as Basal Cell Carcinoma (BCC), Squamous Cell Carcinoma (cSCC), glioma, such as a glioblastoma, or sarcoma, such as soft-tissue sarcoma. The cancer may be metastatic or non-metastatic. The cancer may be recurrent or non-recurrent, such as recurrent glioblastoma.

Where the cancer to be treated with the pharmaceutical preparation of the invention is a glioma, such as a glioblastoma, the pharmaceutical preparation is preferably administered together with lomustine and, optionally, a further chemotherapy. Details of such a therapy are disclosed in WO2021/234178, which is hereby incorporated by reference in its entirety. Thus, the pharmaceutical preparation of the invention may be for use in a method of treating a glioma, such a glioblastoma, in a patient, wherein the method further comprises administering lomustine and optionally, a second chemotherapy, such as temozolomide, to the patient.

Where the cancer to be treated with the pharmaceutical preparation of the invention is a skin cancer, the pharmaceutical preparation is preferably administered together with L19-IL2. The L19-IL2 preferably comprises or consists of the sequence set forth in SEQ ID NO: 14. Details of such a therapy are disclosed in WO2013/045125, which is hereby incorporated by reference in its entirety. Thus, the pharmaceutical preparation of the invention may be for use in a method of treating skin cancer, such as a malignant melanoma or non-melanoma skin cancer, but preferably a malignant melanoma, in a patient, wherein the method further comprises administering L19-IL2 to the patient. Administration may be by injection at the tumor site, in particular, by intra-tumoral injection. Most preferably, the dose of L19-IL2 is 2.17 mg. The dose of L19-IL2 can alternatively be stated in international units (III). 2.17 mg L19-IL2 equates to 13 million IU L19-IL2. In the case of intra-tumoral injections, patients may be treated with 13 million IU of L19-IL2 corresponding to 2.17 mg of L19-IL2 and with 400 pg of L19-TNFa once weekly for one to four weeks, e.g., for one, two, three or four consecutive weeks. The dose may be administered as a single intra-tumoral injection, or a dose may be divided into multiple intratumoral injections, which are administered to the same tumor. Alternatively, in certain cases the dose may halved to be 1.08 mg of L19-IL2 and 200 pg of L19-TNFa.

The features disclosed in the foregoing description, or in the following claims, or in the accompanying drawings, expressed in their specific forms or in terms of a means for performing the disclosed function, or a method or process for obtaining the disclosed results, as appropriate, may, separately, or in any combination of such features, be utilised for realising the invention in diverse forms thereof. While the invention has been described in conjunction with the exemplary embodiments described above, many equivalent modifications and variations will be apparent to those skilled in the art when given this disclosure. Accordingly, the exemplary embodiments of the invention set forth above are considered to be illustrative and not limiting. Various changes to the described embodiments may be made without departing from the spirit and scope of the invention.

For the avoidance of any doubt, any theoretical explanations provided herein are provided for the purposes of improving the understanding of a reader. The inventors do not wish to be bound by any of these theoretical explanations.

Any section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

Throughout this specification, including the claims which follow, unless the context requires otherwise, the word “comprise” and “include”, and variations such as “comprises”, “comprising”, and “including” will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. It must be noted that, as used in the specification and the appended claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. Ranges may be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by the use of the antecedent “about,” it will be understood that the particular value forms another embodiment. The term “about” in relation to a numerical value is optional and means for example +/- 10%.

Examples

Example 1 - Method for producing a preparation of L19-TNFa comprising a low percentage glycosylated L19-TNFa

1. 1 General description of manufacturing process and process controls

L19-TNFa is a recombinant fusion protein stably expressed in in Chinese Hamster Ovary host cells (CHO-K1), cultured in bioreactors using a semi-continuous fermentation approach. L19- TNFa is recovered from the harvested medium by several chromatographic purification steps. The manufacturing process of L19-TNFa Drug Substance can be divided into two upstream phases and two downstream phases.

- Upstream 1 (phase A): inoculum preparation

- Upstream 2 (phase B): fermentation - Downstream 1 (phase C): capture purification by affinity chromatography

- Downstream 2 (phase D): polishing and formulation by gel filtration, cation exchange chromatography, buffer exchange, anion exchange chromatography, nanofiltration and a final concentration.

Both upstream phases are dedicated to cell culturing, whereas the downstream phases are for the recovery of L19-TNFa from the harvested medium by chromatographic purification. Figure 1 shows a summarized process flow chart of the L19-TNFa production. The upstream and downstream processes are reported below.

1.1.1 Upstream process

The Upstream process to produce L19-TNFa can be divided into two phases: Upstream 1 (phase A; inoculum preparation) and Upstream 2 (phase B; fermentation). Both phases can be divided into a number of steps:

Upstream 1 (Phase A)

EX-CELL CD CHO medium (provided by SAFC a unit of Sigma Aldrich; catalogue number 14361C-1000ML) with 6 mM L-Glutamine allows the cultivation or adaptation of recombinant CHO cells in a small scale (flasks). This medium was used in Upstream Phase 1 , supplemented with 20 mM HEPES buffer when used in Roller Bottles.

Upstream 2 (Phase B)

EX-CELL CD CHO medium with 6 mM L-Glutamine was also used for larger scale bioreactor fermentation in phase Upstream 2. Additionally, the feed supplement BalanCD CHO Feed 3 was added to the cell culture of L19-TNFa in phase Upstream 2. Furthermore, glucose 30% w/v solution was added to the cell culture to reach a concentration of 4 g/L glucose when the glucose concentration dropped below 2 g/L.

Upstream 1 (phase A): Inoculum preparation for fermentation

L19-TNFa cell culture began with the thawing of a master cell bank vial suitable for the production of L19-TNFa. The cell culture was first amplified in T75/T150 square flasks containing 15/30 ml of cell culture in standard conditions (37°C, 5% CO2) and then expanded in 2 L roller bottles (37°C, 1.2 rpm) containing 250 ml of cell culture. EX-CELL CD CHO medium, supplemented with 6 mM Glutamine (6-XF), was used for square flask cell culturing. For cell culturing in roller bottles the same medium supplemented with 20 mM HEPES buffer (6-XRG) was used. The cell culture in square flasks and roller bottles was split every 3-4 days as detailed below. Amplifications were carried out at variable dilutions, to reach a viable cell concentration in the range of 100.000-150.000 cell/ml after dilution with fresh medium. To achieve a cell inoculum for bioreactor fermentation, the roller culture was expanded up to a total volume of 9 L (36 roller bottles) and to a cell concentration >500.000 cells/ml with a viability > 60 %.

At the end of the amplification, the cell suspension was transferred into a sterile bag using a peristaltic pump. The sealed bag was then connected to the bioreactor using a steam-in-place stainless steel connector. Upstream 2 (phase B): Fermentation

Fermentation took place in a Biostat C 30 bioreactor (B. Braun). The bioreactor was equipped with a gas mixer with 4 exits and a ring sparger aeration system. The pH was controlled through a CChflow and calibrated by addition of a sodium carbonate solution. The stirrer was a single marine impeller. The temperature was controlled by a water jacket connected to a steam heat exchanger. The working volume was 30 L, the total volume of the vessel was 42 L. The semi- continuous fermentation process started when a portion of cell culture, obtained at the end of the Upstream 1 phase, was added to fresh medium in the bioreactor (inoculum). The fermentation of a single batch may take 27 - 48 days, according to the viability of the cell culture and the production requirements. Two twin bioreactors Biostat C 30 were used for fermentation in a semi-continuous approach in which each bioreactor was split every 6-7 days to harvest the cell culture. The harvested volume depended on the viable cell concentration determined before harvesting. To start each new fermentation cycle with a viable cell concentration of 100.000- 200.000 cells/ml, enough cell culture was retained in the bioreactor to reach a final working volume of 27 L by adding fresh medium. As the process was performed in two twin Biostat C 30 reactors, both bioreactors were concurrently inoculated with portions of the cell culture obtained at the end of Upstream 1 , running the semi-continuous fermentation simultaneously. The general fermentation scheme is outlined in Figure 2. The operational steps of the Upstream 2 (phase B) process are summarized below.

¹ Steps 112-410 to LI2-655 are performed according to the viability of the cell culture and production requirements. Therefore, the fermentation in the bioreactors may be terminated after the 3 ^rd , 4 ^th , 5 ^th or 6 ^th harvesting.

Inoculation of the bioreactor The bioreactor inoculum reaches a viable cell concentration in the range of 100.000-200.000 viable cells/ml at the beginning of the fermentation. The necessary dilution of medium and cell culture was calculated to reach a total working volume of 27 L. Before inoculation, the bioreactor was aseptically filled with the required quantity of culture medium (EX-CELL CHO Medium + 6 mM Glutamine). The medium was transferred into the bioreactor vessel through the steam-in-place sterilized addition port device, passing a 0.22pm filter with help of a peristaltic pump. The required quantity of cell culture, corresponding approximately to 1/4 of total fermentation volume, was then inoculated into the bioreactor vessel to start the fermentation process. The aseptic addition of the inoculum was performed by gravity through the steam-in- place sterilized addition port device of the bioreactor. Fermentation Temperature

The temperature was kept at 37°C ± 0.01 °C during the whole fermentation process.

Culture pH

During fermentation the culture pH was kept stable at a pH of 7.20 ± 0.02 by a flow of CCh for acid corrections (partial pressure: 1 .25 atm) and by calibrated additions of a Na2COs 20 % w/v solution for basic correction. Acid or base additions were regulated automatically by bioreactor management software through a gas mixer device (CO2 addition) and a system peristaltic pump (Sodium Carbonate addition).

Dissolved Oxygen (pO )

The concentration of dissolved oxygen was maintained constant at 25% ± 2% air saturation value with a flow of air, nitrogen, and oxygen. The relative ratios of these three different gases were controlled by the fermentation management software through the gas mixer device of the bioreactor. Partial pressure of each: 0,95 atm for oxygen, nitrogen and air.

Aeration and stirring

The cell culture was aerated through a ring sparger diffusion system. The gas mixture comes out of the sparging device located on the base of the fermentation vessel through 1 mm holes. Bubbles are pushed down by the agitation system (single marine impeller) to increase the contact time of the gas mixture with the cell culture, thereby improving gas solubility. The impeller rotates at 88 rpm ± 5 during the first 24 hours of fermentation, then the rate is increased to 100 rpm ± 5 until the harvesting of the cell culture. The Airflow (gas mixture flow rate) set point is 1 ,0 < flow < 1,3 slpm ± 0,05 during the first 24 hours of every fermentation cycle, then the value is increased to 1 ,3 < flow < 1,5 slpm ± 0,05 until the harvesting of the cell culture.

Semi continuous fermentation and harvesting volume

The fermentation process started after inoculation with the pool of roller bottles from Upstream 1 (phase A) with an initial working volume of 27 L. During each fermentation cycle, the cell culture was fed on day 1, 3 and 5 with the feed (5 % total of the initial volume) as described in section Upstream 2 (phase B). The glucose concentration was regulated in order to reach 4 g/L of glucose concentration in the bioreactor. The fermentation was interrupted cyclically for intermediate harvesting of the cell culture after 6-7 days, harvesting about 30 L of cell culture, replaced with fresh medium to obtain a cell concentration in the range of 100.000-200.000 viable cells/ml at the beginning of each new fermentation cycle. At least 3 and up to a maximum of 7 splitting cycles were performed, depending on the viability of the cell culture and the production requirements. During the fermentation the cell culture was sampled aseptically daily. At the end of the 3 ^rd , 4 ^th , 5 ^th , 6 ^th or 7 ^th cycle, the fermentation process was terminated and the entire cell culture in the bioreactor was harvested. A complete fermentation process therefore lasted a total of 27 to 48 days, including equipment washing and sterilization. A total of seven harvests from each bioreactor can be obtained for a total Crude Harvest volume of about 215 L/bioreactor. An average of 8 g of crude product was obtained from a fermentation run. Microfiltration

Harvested material was clarified and sterilized using single-use cartridge filters. The first step of clarification was carried out with a 0.65 pm, 0.6m ² cartridge filter, while further clarification and sterilization was obtained through a combined 0.22 pm, 0.45m ² cartridge filter. The harvested material was loaded into the filters with a peristaltic pump at a flow rate of 1-2 L/min. Process yield was typically >90 %. After filtration 0.5M EDTA solution pH 8.0 was added to the clarified harvest for a final EDTA concentration of 5mM. The pH of the feedstock was adjusted to pH 7.5 to obtain the Clarified Bulk. The Clarified Bulk can be stored at 2-8 °C for up to two months until the next purification step.

1.1.2 Downstream process

The downstream process for the purification of L19-TNFa included a series of chromatographic and (dia)filtration steps. The process is divided into Downstream 1 (phase C) and Downstream 2 (phase D), according to the following scheme: a) Protein A affinity chromatography (Downstream 1) b) Gel filtration (for buffer exchange) (Downstream 2) c) Cation exchange chromatography (Cl EX) (Downstream 2) d) Gel filtration (for buffer exchange/formulation) (Downstream 2) e) Anion exchange chromatography (Al EX) (Downstream 2) f) Nanofiltration (for virus removal) (Downstream 2) g) Diafiltration (for concentration) (Downstream 2) h) Adding of Tween20 (for formulation completion) (Downstream 2).

Downstream 1 (phase C)

The Downstream 1 process (phase C) consists of an affinity chromatography step using the rmp Protein A Sepharose Fast Flow, a recombinant multipoint ligand protein A resin. Multiple chromatographic runs were performed to process the entire amount of Clarified Bulk obtained at the end of the Upstream process. At the end of this step, the Semi-processed material was then purified in the Downstream 2 process or was otherwise stored at -80 °C for up to 60 days, after addition of 1.5 % w/v glycerol. The purification scheme of Downstream 1 is shown below.

Buffer preparation

Each buffer was prepared in a stainless-steel tank under mechanical agitation, and the pH was adjusted with 5 M NaOH or 5 M HCI. Buffers were then sterilized by filtration and transferred into sterile bags or bottles with the aid of a peristaltic pump. Buffers were stored at 2-8 °C and used within one month.

Affinity Chromatography on Protein A (steps from D1-10 to D1-50)

The Protein A affinity chromatography was performed using the rmp Sepharose Fast Flow resin packed in a Vantage A2-180 column. The resin is a low leakage, non-mammalian-based affinity resin designed for high purity separation of monoclonal and polyclonal antibodies. The base matrix, Sepharose 4 Fast Flow, is a highly cross-linked, 4 % agarose derivative, containing five antigen binding domains. The scFv (L19) moiety of L19-TNFa fusion protein binds to protein A immobilized on the resin. The volume of resin packed in the Vantage A2-180 column differed according to the Table below.

The volume of resin depended on the amount of sample to be purified; usually 1 ml of resin was used to purify up to 4.3 mg of L19-TNFa.

Column sanitization and equilibration

Before sample loading, the column, resin and flow lines of the chromatographic system were sanitized in place with a solution of 0.1 M acetic acid 120% ethanol. The contact time was at least 60 minutes. After sanitization, the system was equilibrated with 2CV PBS pH 7.50 (NaH2PO4'2H2O 50mM; NaCI 150mM, EDTA 5mM, NaOH 50mM), at a flow rate between 85 and 255 ml/min (corresponding to 20 and 60 cm/h). Sample loading

After sanitization, the Clarified Bulk was loaded in fractions of approximately 80L for each run, containing ca. 3800-4300 mg of L19-TNFa. Loading was at room temperature at a flow rate between 100 and 200 ml/min, corresponding to 24 and 48 cm/h, which allowed for a contact time of at least 8 minutes. The sample loading lasted approx. 14 hours.

Sample purification

Once the Clarified Bulk was loaded, the column was washed sequentially with the following buffers:

- 10 CV PBS/NaCI (NaH ₂ PO ₄ '2H ₂ O 50mM; NaCI 650mM, EDTA 5mM, NaOH 53mM, pH

7.50);

- 5 CV PBS pH 7.50 (NaH ₂ PO ₄ 2H ₂ O 50mM; NaCI 150mM, EDTA 5mM, NaOH 50mM).

The L19-TNFa fusion protein was then eluted from the column with TEA 10mM pH 11.50 (TEA 10mM, EDTA 5mM, pH 11.50). Samples were eluted in a single fraction, ranging between 4 and 5 CV. The typical yield of this chromatographic step was higher than 90 %. The chromatographic run was conducted by monitoring the A ₂ sonm, the conductivity and the pH. The eluted sample (the Semi-processed material) was then dissolved in TEA 10mM pH 11.50 (TEA 10mM, EDTA 5mM, pH 11.50) and incubated at room temperature for viral inactivation for 1 hour. The O.D. (A ₂ sonm) at this stage ranged between 0.470 and 1.460.

The Semi-processed material was either processed in the next purification step, in the Downstream 2 process or was stored at -80 °C, after addition of 1 .5 % w/v glycerol, for a maximum period of 60 days.

Downstream 2 (phase D)

The Downstream 2 process (phase D) consisted of a series of chromatographic and (dia)filtration steps, to purify and polish L19-TNFa. The Purified Bulk, obtained at the end of this process, represents the Drug Substance. The purification scheme of Downstream 2 is shown below.

Gel Filtration by Sephadex G25 (buffer exchange) (steps D2-10 to D2-40)

System preparation

The semi-processed material (obtained from Downstream 1) underwent buffer exchange by gel filtration performed using the cross-linked dextran Sephadex G25 Fine resin packed in a

Vantage A2-130 column. The volume of resin packed in the Vantage A2-130 column varied according to the Table below. The volume of resin used depended on the amount of protein to be processed; usually 1 ml of resin is used to purify up to 0.55 mg of L19-TNFa.

Column sanitization and equilibration

Before sample loading, the column, resin and flow lines of the chromatographic system were sanitized in place with a solution of NaOH 0.2M. After sanitization, the system was equilibrated with 2 CV “Buffer phosphate 5mM” solution (NabhPO^FhO 5mM, pH 6.50), at a flow rate between 100 and 300 ml/min (corresponding to 45 and 135 cm/h). Sample loading

After sanitization, the semi-processed material was loaded at a flow rate between 100 and 300 ml/min (corresponding to 45 and 135 cm/h). The semi-processed material was loaded in several fractions (generally 3-4 fractions), so that the load does not exceed the column’s capacity (20 % of column volume).

Sample elution

L19-TNFa was eluted (as desalted semi-processed material) with 0.6 CV “Buffer phosphate 5mM” solution (NaH2PO4'2H2O 5mM, pH 6.50) at a flow rate between 100 and 300 ml/min (corresponding to 45 and 135 cm/h). The buffer exchange by gel filtration included a dilution of approx. 2-3 fold. The typical yield of this chromatographic step was 65-80 %. The chromatographic run was conducted by monitoring A280nm, conductivity and pH. The desalted semi-processed material can be stored at 2-8 °C for a maximum period of 72 hours.

Cation Exchange Chromatography (Cl EX) (steps from D2-50 to D2-60)

System preparation

The cation exchange chromatography (Cl EX) was performed using a CHT ceramic hydroxyapatite type II (40pm) resin, packed in a Vantage A2-60 column. Sets of five calcium doublets (C-sites) and pairs of hydroxyl-containing phosphate triplets (P-sites) were arranged in a repeating geometric pattern. The volume of resin packed in the column varied according to the Table below.

The volume of resin used depended on the amount of sample to be purified; usually 1 ml of resin is used to purify up to 2.7 mg of L19- TNFa.

Column sanitization and equilibration

Before the sample loading, the column, resin and flow lines of the chromatographic system were sanitized with a solution of NaOH 1M. The contact time was at least 60 minutes. After sanitization, the system was equilibrated with 10 CV “Buffer A phosphate” solution (NaH2PO4'2H2O 10mM, pH 6.50), at a flow rate between 95 and 235 ml/min (corresponding to 200 and 500 cm/h). A Sartobind Q cartridge (0.250 m ² filtration area) was installed in series before the CHT column. The Sartobind Q cartridge was separately activated with NaCI 1M, sanitized with NaOH 1M (60 minutes at least) and equilibrated with “Buffer phosphate 5mM” solution (NaH ₂ PO ₄ -2H ₂ O 5mM, pH 6.50).

Sample loading

After column sanitization, the desalted semi-processed material was loaded onto the system Sartobind Q-CHT column. The loading was performed at a flow rate between 95 and 235 ml/min (corresponding to 200 and 500 cm/h), which allowed for a contact time with the CHT resin of at least 2.5 minutes.

Sample elution

After loading, the CHT column was washed with 2 CV “Buffer A phosphate” solution (NaH ₂ PO4'2H ₂ O 10mM, pH 6.50). Then the L19-TNFa fusion protein was eluted from the column by a linear gradient, obtained by introducing progressively 40 CV “Buffer B phosphate” solution (NaH ₂ PO4'2H ₂ O 10mM, NaCI 1M, pH 6.50) from 0 to 100 %, at a flow rate of 200 ml/min. The typical yield of this chromatographic step was around 70 %. The chromatographic run was conducted by monitoring A ₂ sonm, conductivity and pH. The purity of the Purification Intermediate 1, obtained at the end of the cation exchange chromatography, was higher than 98% for L19-TNFa total and higher than 95% for L19-TNFa homotrimer. The Purification Intermediate 1 can be stored at 2-8 °C for a maximum period of 48 hours.

Gel Filtration by Sephadex G25 (buffer exchange/formulation) (step D2-70)

System preparation

The third operative step of Downstream 2 is the formulation of Purification Intermediate 1, performed through a buffer exchange by gel filtration chromatography. The Purification Intermediate 1 was processed to transfer the fusion protein L19-TNFa into its formulation buffer. The gel filtration was performed using the cross-linked dextran Sephadex G25 Fine resin, packed in a Vantage A2-130 column. The volume of resin which was packed in the Vantage A2- 130 column varied according to the Table below.

The volume of resin used depended on the amount of protein to be processed; usually 1 ml of resin is used to purify up to ca. 0.30 mg of L19-TNFa. Column sanitization and equilibration

Before sample loading, the column, resin and flow lines of the chromatographic system were sanitized in place with a solution of NaOH 0.2M. The contact time was at least 60 minutes. After sanitization, the system was equilibrated with 2 CV “Formulation buffer L19-TNFa without Tween20” (NaH ₂ PO ₄ '2H ₂ O 15mM, Na ₂ HPO ₄ -2H ₂ O 10mM, KCI 1.5mM, Mannitol 75mM, NaCI 30mM, Glycerol 1 % w/v, EDTA 5mM, pH 8.00), at a flow rate between 100 and 300 ml/min (corresponding to 45 and 135 cm/h).

Sample loading

After sanitization, the Purification Intermediate 1 was loaded at a flow rate between 100 and 300 ml/min (corresponding to 45 and 135 cm/h). The Purification Intermediate 1 was loaded in several fractions (generally 5 f), as the load should not exceed the column’s capacity (30 % of column volume). Each fraction loading was followed by elution.

Sample elution

L19-TNFa was eluted as Purification Intermediate 2 with 0.6 CV “Formulation buffer L19-TNFa without Tween20” (NaH ₂ PO ₄ -2H ₂ O 15mM, Na ₂ HPO ₄ -2H ₂ O 10mM, KCI 1.5mM, Mannitol 75mM, NaCI 30mM, Glycerol 1 % w/v, EDTA 5mM, pH 8.00) at a flow rate between 150 and 250 ml/min (corresponding to 70 and 113 cm/h). The buffer exchange by gel filtration included a dilution of around 1.5-fold. The typical yield of this chromatographic step was around 95 %. The entire process generally lasted ca. 2 hours. The chromatographic run was conducted by monitoring A ₂ 80nm, conductivity and pH. The Purification Intermediate 2 was then directly processed in the next purification step of anion exchange chromatography.

Anion exchange chromatography (AIEX) by Sartobind Q (step D2-80)

The Purification Intermediate 2 was processed by anion exchange chromatography (AIEX), using a single use Sartobind Q capsule of 0.250 m ² filtering area. Before sample loading, the capsule was activated flushing 200ml NaCI 1 M; then the capsule was sanitized by flushing NaOH 1 M, for at least 60 minutes, and equilibrated by flushing “Formulation buffer L19-TNFa without Tween20” (NaH ₂ PO ₄ -2H ₂ O 15mM, Na ₂ HPO ₄ -2H ₂ O 10mM, KCI 1.5mM, Mannitol 75mM, NaCI 30mM, Glycerol 1 % w/v, EDTA 5mM, pH 8.00) at a flow rate between 150 and 300 ml/min. The Purification Intermediate 2 was loaded at a flow rate between 150 and 250 ml/min. The L19-TNFa protein is positively charged (pH 8.0), therefore the Sartobind Q capsule was used in a flow through mode, collecting L19-TNFa directly into a container as Purification Intermediate 3. Possible contaminants negatively charged (such as residual DNA), were removed by binding to the Sartobind Q matrix. The typical yield of this chromatographic step was higher than 95 %. The Purification Intermediate 3 can be stored at 2-8 °C for a maximum period of 48 hours, before the next purification step of nanofiltration. Nanofiltration (virus removal) (step D2-90)

The Purification Intermediate 3 was processed for viral clearance by nanofiltration, using the Viresolve ProModus 1.1 or Viresolve ProModus 1.2 filters. The ProModus 1.1 or 1.2 can be used, with 0.017 and 0.070 m ² filtering area, respectively. The membrane nominal cut-off is ca. 20nm. Before use, the cartridge was wetted by flushing water for injection for 10 minutes at a constant pressure of 1.8 bar, it was then sanitized by flushing NaOH 0.5M for at least 60 minutes at the same pressure. After sanitization, the cartridge was equilibrated with “Formulation buffer L19-TNFa without Tween20” (NaH ₂ PO ₄ '2H ₂ O 15mM, Na ₂ HPO ₄ -2H ₂ O 10mM, KCI 1.5mM, Mannitol 75mM, NaCI 30mM, Glycerol 1 % w/v, EDTA 5mM, pH 8.00) flushed at a constant pressure of 3 bar for 5 minutes. After equilibration, the Purification Intermediate 3 was loaded at a flow rate at ca. 300 ml/min (for Viresolve ProModus 1.2 filter). The L19-TNFa recovery after nanofiltration was usually higher than 99 %. At the end of nanofiltration the Purification Intermediate 4 was obtained. If the optical density was lower than 0.530, the Purification Intermediate 4 was concentrated in the next Downstream 2 step. Otherwise, it was added directly with 0.01 % v/v Tween20 to complete the Drug Substance formulation, obtaining the Purified Bulk.

Dia filtration (for concentration) (step D2-100)

If the optical density was lower than 0.530, the Purification Intermediate 4 was concentrated by diafiltration, to adjust its optical density to a suitable range comprised between 0.530 and 0.580. The diafiltration was performed using the Sartocon Slice cassette system. The system was sanitized by fluxing NaOH 1M for at least 60 minutes, before being rinsed with water for injection and equilibrated with “Formulation buffer L19-TNFa without Tween20” (NaH ₂ PO4'2H ₂ O 15mM, Na ₂ HPO4'2H ₂ O 10mM, KCI 1.5mM, Mannitol 75mM, NaCI 30mM, Glycerol 1 % w/v, EDTA 5mM, pH 8.00), at a flow rate of ca. 50 ml/min. When the equilibration was complete, the Purification Intermediate 4 was loaded into the system. The Purification Intermediate 4 was continuously flushed through the Sartocon cassette. L19-TNFa was retained and returned to the starting bottle “Retentate”, whereas the formulation buffer, which passes the membrane, was discarded in the bottle “Permeate”. Since the buffer was not replaced, the L19-TNFa concentration progressively increased. During the concentration, the Purification Intermediate 4 volume decreased approx. 2-4 fold, according to the starting optical density. The process was controlled by monitoring the optical density value. When it reached the established range 0.530- 0.580, the concentration process was stopped. The yield of this operative step was generally higher than 99%. The obtained Purification Intermediate 5 can then be directly added with 0.01% v/v Tween20 to complete the Drug Substance formulation. Addition of 0.01 % v/v Tween20 (step D2-110)

The Drug Substance formulation is obtained by adding 0.01 % v/v Tween20. A stock solution of Tween20 10 % v/v was added in a suitable quantity, to reach the final content 0.01 % v/v Tween20. Tween20 was added to the Purification Intermediate 4 at the end of the nanofiltration, if the optical density was already in the range 0.530-0.580, otherwise it was added to the Purification Intermediate 5, after the completion of the concentration process. The addition of 0.01 % v/v Tween20 completed the Downstream 2 process. The Purified Bulk (Drug Substance) can be stored at 2-8 °C for a maximum period of 7 days or at -80 °C for up to 1 month.

Example 2 - Quality Control of GMP lots

Purity of the good manufacturing process (GMP) lots prepared according to Example 1 were characterized using several quality control analyses, including ELISA, SDS-PAGE, Size Exclusion Chromatography, A280 measurements, productivity, bioactivity and immunoreactivity assays, osmolality, viral testing, visual appearance etc. The protocol used for 2D-SDS-PAGE analysis, is reported below.

2. 1 2D-SDS-PAGE

Each sample was analyzed in a bidimensional electrophoresis assay using a precast system (ZOOM® IPGRunner™). The assay was performed by adopting two ranges of pH for iso-electric focusing:

1) pH 3.0 - 10.0

2) pH 6.0 - 10.0.

In (1), the pH range is wide enough to underline eventual contaminants while in (2) the pH range separation is smaller but still includes the isoelectric point of the sample (pH = 7.7). Staining was performed with Blue Coomassie R-250. The results are reported in Figure 3.

Example 3 - Mass spectrometry analysis of GMP lots

3. 1 Direct Infusion HR-MS for intact mass analysis

100 pg of L19-TNFa samples were desalted using C18-purification before analysis (Micro Spin Columns, Harvard Apparatus). Direct infusion was performed on an Orbitrap Q-Exactive coupled to an Ion Max ESI source. The following parameters were used: syringe flow rate 4 pL/min, capillary voltage 3.0 kV, in source induced dissociation 40 eV, sheath gas 4 units, capillary temperature 300 °C, S-lens RF level 90, resolution 17500 (FWHM at 200 m/z), AGC target 5 x 10 ⁴ , microscan 10, mass range 500-3000 m/z, and maximum injection time 200 ms. Raw spectra were then analyzed to quantify relative amounts of glycosylated and nonglycosylated L19-TNFa of the total L19-TNFa in the preparation. Quantification was achieved by summing the intensities of the peaks corresponding to glycosylated or non-glycosylated L19- TNFa, respectively, and the percentage was calculated using the equation below.

Intensity of x , Intensities ^x ioo

Where x is the sum of the intensities corresponding to non-glycosylated or glycosylated L19- TNFa, respectively. The results of this analysis are shown in Figures 4 and 5.

3.2 MS/MS analysis for glycopeptide analysis

7.5 pg of L19-TNFa were resuspended in urea 1M dissolved in NH4HCO3 solution at a final pH = 8. Protein was reduced with TCEP for 15 min at RT followed by 45 min at 65°C and alkylated with lodoacetamide (IAA) for 1 hour in the dark. L19-TNFa was then digested by trypsin (enzyme-protein ratio 1 :60) at 37°C overnight. After digestion, the sample was acidified with 10% formic acid and then subjected to C18 purification and desalting (Macro Spin Columns, Harvard Apparatus). 500 ng of the resulting peptides were then subjected to HPLC-MS/MS analysis. All samples were analysed on an Orbitrap Q-Exactive mass spectrometer coupled to an EASY nanoLC 1000 system via a Nano Flex ion source. Chromatographic separation was carried out on an Acclaim PepMap RSLC column (50 pm x 15 cm, particle size 2 pm, pore size, 100 A), using 40 min linear gradient with 5-35% solvent B (0.1% formic acid in acetonitrile) at a flow rate of 300 nL/min. Ionization was carried out in positive ion mode, with 2 kV of spray voltage, 250°C of capillary temperature, 60 S-lens RF level. The mass spectrometer was working in a data-dependent mode. MS1 scan range was set from 350 to 1650 m/z, the 10 most abundant peptides were subjected to HCD fragmentation with NCE of 25. A dynamic exclusion was set at 10 seconds. Raw files were processed with Proteome Discoverer 1.4. Database searches were performed using Sequest as search engine using a FASTA file containing our protein of interest, the mus musculus reference proteome and additional contaminants (human keratin isoforms, bovine serum albumin and ProteinA from Staphylococcus Aureus). Carbamidomethylation of cysteines was set as a fixed modification while oxidation of methionine and the O-glycosylation (HexNAciHexiNeuAci) were set as variable modifications. Trypsin was set as cleavage specificity, allowing a maximum of 2 missed cleavages. Data filtering was performed using percolator, resulting in 1% false discovery rate (FDR). The results of this analysis are reported in Figures 6 and 7. Sequence Listing

Amino acid sequence of L19 CDRs

L19 CDR1 VH -SFSMS (SEQ ID NO: 1)

L19 CDR2 VH -SISGSSGTTYYADSVKG (SEQ ID NO: 2) L19 CDR3 VH -PFPYFDY (SEQ ID NO: 3)

L19 CDR1 VL -RASQSVSSSFLA (SEQ ID NO: 4)

L19 CDR2 VL -YASSRAT (SEQ ID NO: 5)

L19 CDR3 VL -QQTGRIPPT (SEQ ID NO: 6)

Amino acid sequence of the L19 VH domain (SEQ ID NO: 7)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGT TYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSS

Amino acid sequence of the linker between VH and VL (SEQ ID NO: 8) GDGSSGGSGGAS

Amino acid sequence of the L19 VL domain (SEQ ID NO: 9)

EIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRAT GIPDRFS

GSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

Amino acid sequence of the L19 scFv (SEQ ID NO: 10)

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGT TYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDG SSGGS

GGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYAS SRATGIP

DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIK

Amino acid sequence of the soluble form of the extracellular domain of human TNFa (huTNFa)

(SEQ ID NO: 11).

VRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYL IYSQVL

FKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYL GGVFQLEK

GDRLSAEINRPDYLDFAESGQVYFGIIAL

Amino acid sequence of the linker between scFv and TNFa (SEQ ID NO: 12) EFSSSSGSSSSGSSSSG Amino acid sequence of the L19-huTNFa conjugate (SEQ ID NO: 13)

Serine 257 is shown in bold, italics and underlined

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGT TYYAD

SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDG SSGGS GGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRA TGIP

DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSSSGSS SSGSSSS GVRSSSRTPSDKPVAHVVANPQAEGQLQWLNRRANALLANGVELRDNQLVVPSEGLYLIY SQ VLFKGQGCPSTHVLLTHTISRIAVSYQTKVNLLSAIKSPCQRETPEGAEAKPWYEPIYLG GVFQL EKGDRLSAEINRPDYLDFAESGQVYFGIIAL Amino acid sequence of the L19-IL2 conjugate (SEQ ID NO: 14)

Threonine 256 is shown in bold, italics and underlined

EVQLLESGGGLVQPGGSLRLSCAASGFTFSSFSMSWVRQAPGKGLEWVSSISGSSGT TYYAD SVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKPFPYFDYWGQGTLVTVSSGDGSSG GS GGASEIVLTQSPGTLSLSPGERATLSCRASQSVSSSFLAWYQQKPGQAPRLLIYYASSRA TGIP DRFSGSGSGTDFTLTISRLEPEDFAVYYCQQTGRIPPTFGQGTKVEIKEFSSSSGSSSSG SSSS

GAP7SSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHL QCLEEELK PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFLNRWITF CQSIIST LT