Combination of an immunocytokine comprising targeted il2 and a JAK inhibitor

Abstract

The invention relates to a combination of (i) a targeted immunocytokine comprising interleukin-2 (IL2) and a targeting entity that binds to fibronectin, and (ii) a Janus Kinase (JAK) inhibitor.

Description

[0001] Combination of an immunocytokine comprising targeted IL2 and a JAK Inhibitor

[0002] Reference to sequence listing submitted as a compliant xml 1.0 format file (.xml)

[0003] Pursuant to the EFS-Web legal framework and 37 CFR §§ 1.821-825 (see MPEP § 2442.03(a)), Rule 30 EPC, and § 11 PatV, an electronic sequence listing compliant with WIPO standard ST.26 in the form of an XML 1.0 format file is submitted concurrently with the instant application, and the entire contents of the sequence listing are incorporated herein by reference. For the avoidance of doubt, if discrepancies exist between the sequences mentioned in the specification and the electronic sequence listing, the sequences in the specification shall be deemed to be the correct ones.

[0004] Field of the invention

[0005] The invention relates to the field of immunoconjugates.

[0006] Incorporation by Reference

[0007] All publications, patents, patent applications and other documents cited in this application are hereby incorporated by reference in their entireties for all purposes to the same extent as if each individual publication, patent, patent application or other document were individually indicated to be incorporated by reference for all purposes. In the event that there are any inconsistencies between the teachings of one or more of the references incorporated herein and the present disclosure, the teachings of the present specification are intended.

[0008] Background Cytokines are key mediators of innate and adaptive immunity. Many cytokines have been used for therapeutic purposes in patients with advanced cancer, but their administration is typically associated with severe toxicity, hampering dose escalation to therapeutically active regimens and their development as anticancer drugs. To overcome these problems, the use of ‘immunocytokines’ (i.e. cytokines fused to antibodies or antibody fragments) has been proposed, with the aim to concentrate the immune-system stimulating activity at the site of disease while sparing normal tissues (Neri & Bicknell, 2005). However, genetically fusing a cytokine to an antibody or to an antibody fragment creating an “immunocytokine”, does not always result in an immunocytokine that retains the ability to target the tumor of the antibody. For example, in certain Interleukin-7 fusions (Pasche et al. (2011) J Biotechnology, 154, 84- 92) the tumor targeting was completely abrogated, while in certain GM-CSF fusions (Kaspar et al. (2007) Cancer Res, 67, 4940-4948) the tumor targeting ability was found to be dose dependent.

[0009] IL2 is a small 15-kDa cytokine with pleiotropic effects on the immune system. Low doses of IL2 preferentially bind to the trimeric IL2 receptor (IL2R), consisting of IL2Ra (CD25), IL2RP (CD122), and the common gamma chain (CD132), which is mainly expressed on immunosuppressive regulatory T (Treg) cells. Trimeric IL2Rs are also referred to as high- affinity IL2Rs because their affinity for IL2 is about 10-100 times higher than that of dimeric IL2Rs. Once the limited amounts of trimeric IL2Rs on these cells are saturated, IL2 also very efficiently associates with and stimulates dimeric IL2Rs, made of CD122 and CD132, that are principally present on resting antigen-experienced (memory) effector T (Teff) and natural killer (NK) cells (Boyman & Sprent (2012) Nat Rev Immunol, 12, 180-190).

[0010] The stimulatory effect of IL2 on Teff and NK cells motivated trials of high-dose IL2 for the treatment of cancer, with recombinant human IL2 (Aldesleukin) becoming the first US Food and Drug Administration approved immunotherapy for the treatment of metastatic renal cell carcinoma (RCC) and metastatic melanoma in 1992 and 1998, respectively (Rosenberg (2014) J immunol, 192, 5451-5458). Based on the results of clinical trials and practical experiences, a two-cycle course of high-dose IL2 administered intravenously (IV) is standard. Each course consists of two 5-day cycles (600,000 lU / kg / dose administered IV over 15 minutes Q8H) separated by a minimum of 9 days. If tolerated, IL2 is given for a maximum of 14 doses per cycle and 28 doses per course (Proleukin® [package insert], Emeryville, Calif: Chiron Corporation; 2000).

[0011] However, just like many other cytokines, the administration of recombinant human IL2 is associated with severe toxicity, hampering its development as an anticancer drug. High-dose IL2 treatments often lead to severe side effects, including fever and chills, hypotension, fatigue and vascular leak syndrome, which in extreme cases can lead to organ failure with lethal consequences (Dutcher et al (2014) J Immunother Cancer, 2, 26). Because of intolerable side effects, most patients do not receive 100% of the planned dosing in a full cycle of IL2 treatment and high dosage treatments are usually reserved to young and physically fit patients only. This dose-escalation limitation led to modest objective responses in the clinic where only 9.3% of patients with RCC and 4.0% with metastatic melanoma achieved complete responses (CR) on high-dose IL2 monotherapy (Yang et al. (2003) J Clin Oncol, 21, 3127-3132; Atkins et al. (2000) Cancer J Sci Am, 6, S11-S14). These shortcomings of high-dose IL2 treatment motivated the development of improved IL2-based biologic agents with reduced toxicity.

[0012] Initially, administration of high-dose IL2 was associated with mortality rates of up to 4% (Fyfe et al. (1995) Clin Oncol, 13, 688-696). Although mortality rates have decreased substantially, IL2 therapy still causes significant dose-related morbidity (Kammula et al. (1998) Cancer, 83, 797-805). Lower doses, prolonged infusions, and subcutaneous administration have been evaluated as strategies for improving IL2 tolerability, but these regimens have produced response rates lower than those produced with high-dose regimens (Dutcher et al. (1997) Cancer J Sci Am, 3, S73-S78).

[0013] Previously, researchers have attempted to overcome these drawbacks by targeted delivery of IL2 cytokine to the tumour environment through, e.g., conjugation to antibodies specific for antigens associated to cancer growth. These cytokine-antibody conjugates are often referred to as “immunocytokines”, or, more precisely for the current context, “targeted IL2”.

[0014] However, the need to improve the therapeutic index of targeted IL2 remains. For example, it would be highly advantageous to find a way to decrease the IL2 related toxicity while maintaining its anti-cancer efficacy. To overcome the drawbacks associated with IL2 therapy, delivery of IL2 to the tumor site by means of an antibody directed against tumor-associated markers to increase local concentrations of IL2 at the tumour site, as well as reduce toxicities associated with systemic administration of IL2 has been proposed. In particular, the concentration of cytokines at the level of tumour blood vessels is an attractive therapeutic strategy as the tumour neovasculature is more accessible to intravenously administered therapeutic agents than tumour cells, which helps avoid problems associated with the interstitial hypertension of solid tumours. In addition, angiogenesis is characteristic of most aggressive solid tumours. Angiogenesis describes the growth of new blood vessels from existing blood vessels. Tumours can induce angiogenesis through secretion of various growth factors (e.g. Vascular Endothelial Growth Factor). Tumour angiogenesis allows tumours to grow beyond a few millimeters in diameter and is also a prerequisite for tumour metastasis. New blood vessels formed as the result of angiogenesis form the neovasculature of the tumour or the tumour metastases. Targeting IL2 to the neovasculature should allow the immunotherapy of a variety of different tumour types.

[0015] The alternatively spliced extra domain B (ED-B) of fibronectin represent one of the best- characterised markers of angiogenesis and has been reported to be expressed around the neovasculature and in the stroma of virtually all types of aggressive solid tumours (WO97 / 45544). Furthermore, even non-solid cancers, such as leukaemia, may be amenable to treatment by targeting antigens of the neovasculature. WO2011 / 015333 described treating leukaemia, including acute myeloid leukaemia, by targeting the bone marrow neovasculature.

[0016] A human monoclonal antibody specific to this target named L19 has been extensively described (WO1999 / 058570, W02003 / 076469, W02005 / 023318). In addition, immunocytokines based on L19 are currently being investigated in Phase I, Phase II and Phase III clinical trials in patients with cancer. These immunocytokines include several cytokines, comprising IL2.

[0017] L19-IL2 (Darleukin) (W02001 / 062298) an immunocytokine composed of L19 and IL2 has been tested in a variety of therapeutic regimens and combinations thereof for treatment of different types of cancer (W02007 / 115837, W02009 / 089858, WO2013 / 010749, WO2013 / 045125, WO2018 / 115377, WO2018 / 154517, Wagner et al (2008) Clin Cancer Res 14, 4951, Schliemann et al (2009 Blood 113, 2275, Zegers et al (2015) Clin Cancer Res 21, 1151, Cazzamalli et al., (2018) Clin Cancer Res, 24, 3656) with good results. Novel formats of L19-IL2, featuring different arrangements of antibody and IL2 have also been reported by the current applicants (W02020 / 070150)

[0018] The extra domain A (ED-A) of fibronectin is another cancer-related marker. ED-A is a known ligand for the a4pi integrin receptor, ED-A, which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors.

[0019] The applicants of the present invention have generated a human monoclonal antibody specific to this target named F8 which has been extensively described (W02008 / 120101, Villa et al., 2008). In addition, immunocytokines based on F8 are currently being investigated in Phase I, Phase II and Phase III clinical trials in patients with cancer. These immunocytokines include several cytokines, comprising IL2. Similarly to L19-IL2, F8-IL2 has also been tested in a variety of therapeutic regimens and combinations thereof with certain inhibitors (WO2010 / 078945, Frey et al., (2010) J Urol, 184, 2540, Gutbrodt et al., (2013) Sci Transl Med, 5, Wiekowski et al., (2015) Lung Cancer, 9-15, Hutchmaker (2019) Cancer Immunol Res 7, 572, Ziffels et al., (2018) Immunotherapy, 10, 177, Pretto et al (2014) 63, 901, Moschetta et al., (2012) Cancer Res 72, 1814).

[0020] The applicants of the present invention have generated many other targeted immunocytokines such as F8-IL4 (WO2014 / 173570, WO2018 / 069467), L19-IL10 (WO2007 / 128563), F8-IL10 (W02009 / 056268), L19-IL12 (W02006 / 119897, W02013 / 014149, WO2019 / 154986), F8- IL12 (W02013 / 014149) and combinations thereof with certain inhibitors (WO2023 / 131611), F8-VEGF (WO2018 / 224550), F8-IL22 (WO2017 / 009469), F8-IL9 (WO2022 / 018126), L19- INFy (WO2022 / 214664).

[0021] It is one object of the present invention to broaden the scope of therapeutic applications of the above identified immunocytokines.

[0022] It is another object of the present invention to provide new therapeutic options for conditions for which so far no adequate treatment option exists.

[0023] It is another object of the present invention to improve efficacy of a therapy using the above described immunocytokines. Brief description of the Figures

[0024] Figure 1A: Quality control of purified F8-IL2 by SDS-PAGE under non reducing (NR) and reducing (R) conditions (left) and by size exclusion chromatography (right).

[0025] Figure IB: Quality control of purified L19-IL2 by SDS-PAGE under non reducing (NR) and reducing (R) conditions (left) and by size exclusion chromatography (right).

[0026] Figure 2: In vitro bioactivity assay. Mouse CTLL2 cells were incubated in presence of the anti ED-A construct F8-IL2 (Figure 2A) or the anti ED-B construct L19-IL2 (Figure 2B), and different doses of inhibitors. After 72h, cell proliferation was measured with CellTiter 96® AQueous One Solution Cell Proliferation Assay.

[0027] Figure 3: In vitro bioactivity assay. Mouse CTLL2 cells were incubated in presence of F8-IL2 and different doses of inhibitors. After 72h, Interferon-y levels were quantified by ELISA.

[0028] Figure 4: In vitro bioactivity assay. Mouse CTLL2 cells were incubated in presence of F8-IL2 and different doses of inhibitors. After 72h, TNFct levels were quantified by ELISA.

[0029] Figure 5: In vitro bioactivity assay showing equal potency of Upadacitinib on F8-IL2 and L19- IL2. Mouse CTLL2 cells were incubated in presence of InM F8-IL2 or L19-IL2 and different doses of Upadacitinib. After 72h, cell proliferation was measured with CellTiter 96® AQueous One Solution Cell Proliferation Assay.

[0030] Figure 6A: In vivo therapy experiment in tumor-bearing mice receiving the JAK inhibitor Upadacitinib, the targeted IL2 (F8-IL2), or a combination of both. Tumor-bearing mice were randomized in different treatment groups. Tumor volumes were measured daily with a caliper. Black arrows represent treatment injection days.

[0031] Figure 6B: In vivo therapy experiment in tumor-bearing mice receiving the JAK inhibitor Upadacitinib, the targeted IL2 (F8-IL2), or a combination of both. Tumor bearing mice were randomized in different treatment groups. Body weights were measured daily and expressed as percentage of change relative to baseline (before therapy). Black arrows represent treatment injection days.

[0032] Figure 6C: Post-mortem analysis of lungs in tumor-bearing mice receiving the JAK inhibitor Upadacitinib, the targeted IL2 (F8-IL2), or a combination of both. Lungs were weighted 24 hours after sacrifice.

[0033] Figure 6D: Quantification of liver edema. Mice were euthanized 24 hours after the last treatment administration. Livers were weighted to determine the extent of edema. Data represent mean ± SD

[0034] Figure 6E: Quantification of the levels of IFNy in plasma. Mice were euthanized 24 hours after the last treatment administration and IFNy in plasma was quantified by ELISA. Data represent mean ± SD

[0035] Figure 7: Comparative study to measure potency of Upadacitinib and Sunitinib when combined with F8-IL2 (Figure 7A) or L19-IL2 (Figure 7B).

[0036] Figure 8: Chemical structure of Upadacitinib

[0037] Figure 9: Schematic figures of F8-IL2 and L19-IL2

[0038] Figure 10: Comparative study to measure potency of Upadacitinib and Peficitinib when combined with the anti-ED-A construct F8-IL2 (Figure 10A) or the anti-ED-B construct L19- IL2 (Figure 10B).

[0039] Detailed Description of embodiments

[0040] Before the invention is described in detail, it is to be understood that this invention is not limited to the particular component parts of the devices described or process steps of the methods described, as such devices and methods may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting. It must be noted that, as used in the specification and the appended claims, the singular forms "a", "an", and "the" include singular and / or plural referents unless the context clearly dictates otherwise. It is moreover to be understood that, in case parameter ranges are given which are delimited by numeric values, the ranges are deemed to include these limitation values.

[0041] It is further to be understood that embodiments disclosed herein are not meant to be understood as individual embodiments which would not relate to one another. Features discussed with one embodiment are meant to be disclosed also in connection with other embodiments shown herein. If, in one case, a specific feature is not disclosed with one embodiment, but with another, the skilled person would understand that does not necessarily mean that said feature is not meant to be disclosed with said other embodiment. The skilled person would understand that it is the gist of this application to disclose said feature also for the other embodiment, but that just for purposes of clarity and to keep the specification in a manageable volume this has not been done.

[0042] Furthermore, the content of the prior art documents referred to herein is incorporated by reference. This refers, particularly, for prior art documents that disclose standard or routine methods. In that case, the incorporation by reference has mainly the purpose to provide sufficient enabling disclosure, and avoid lengthy repetitions.

[0043] According to aspects of the invention, a pharmaceutical composition, dosage form, combination or kit of dosage forms is provided, comprising

[0044] (a) a recombinant protein comprising

[0045] (i) interleukin-2 (IL2) and

[0046] (ii) a targeting entity which binds to fibronectin, which targeting entity comprises an antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin, or a target binding fragment thereof, and

[0047] (b) a Janus Kinase (JAK) inhibitor, wherein the JAK Kinase inhibitor is Upadacitinib,

[0048] According to embodiments, the antibody binding the extra-domain B (ED-B) or the extradomain A (ED-A) of fibronectin a) comprises a set of three heavy chain and three light chain complementarity determining regions (CDR) selected from

[0049] • SEQ ID NOs: 6, 7, 8, 9, 10, and 11 • SEQ ID NOs: 21, 22, 23, 24, 25, and 26, and / or b) comprises the heavy chain / light chain variable domain (HCVD / LCVD) pairs set forth in the following pairs of SEQ ID NOs:

[0050] • 1 and 3;

[0051] • 13 and 15.

[0052] According to embodiments,

[0053] • the antibody or fragment that binds to fibronectin is provided in a single chain Fv format (scFv) or in a diabody format, preferably comprising the amino acid sequence of SEQ ID NO: 28 or 29

[0054] • the targeted IL2 comprises the amino acid sequence of SEQ ID NO: 5 or 20.

[0055] According to another aspect of the invention, a pharmaceutical composition, dosage form, combination or kit of dosage forms is provided, comprising

[0056] (a) a recombinant protein comprising

[0057] (i) interleukin-2 (IL2) and

[0058] (ii) a targeting entity that binds to fibronectin, and

[0059] (b) a Janus Kinase (JAK) inhibitor wherein optionally the recombinant protein and the Janus Kinase (JAK) inhibitor are provided in a separate dosage form, and wherein optionally the recombinant protein and / or the Janus Kinase (JAK) inhibitor are provided in, or together with, a pharmaceutically acceptable carrier.

[0060] The “recombinant protein” referred to herein is also called “immunocytokine” or “targeted immunocytokine”

[0061] As used herein, the term “Immunocytokine” relates to fusion proteins consisting of a cytokine moiety fused to a targeting entity. Immunocytokine products, which are specific to tumor- associated antigens on the cell membrane, have the potential to bridge tumor cells and certain leukocytes (e.g., T cells, or NK cells), in analogy to what could be achieved by using bispecific antibodies. By contrast, immunocytokines which target tumor-associated extracellular matrix components (e.g., splice isoforms of fibronectin or of tenascin-C), are believed to mainly display a biological activity which results from the high-density anchoring of the cytokine moiety at the site of disease.

[0062] As used herein, the term “targeting entity” relates to a molecule that is able to bind to a given target with high specificity and affinity. Such targeting entities are for example antibodies, or antibody fragments.

[0063] The present inventors have unexpectedly recognised that kinase inhibitors reduce the toxicity of targeted IL2, while leaving its anti-cancer activity unaltered.

[0064] In particular, the present inventors have found that when a targeted immunocytokine comprising interleukin-2 (IL2) is administered in tumor bearing mice in combination with such kinase inhibitors, the targeted IL2 maintains its anti-cancer activity but, at the same time, a remarkable reduction in its toxicity is obtained.

[0065] The term Janus Kinase inhibitor or JAK inhibitor, as used herein, refers to inhibitors of the Janus Kinase (JAK). Janus kinase is a family of intracellular, non-receptor tyrosine kinases that transduce cytokine-mediated signals via the JAK-STAT pathway. JAKs possess two nearidentical phosphate-transferring domains. One domain exhibits the kinase activity, while the other negatively regulates the kinase activity of the first. The four JAK family members are Janus kinase 1 (JAK1), Janus kinase 2 (JAK2), Janus kinase 3 (JAK3) and Tyrosine kinase 2 (TYK2).

[0066] Since members of the type I and type II cytokine receptor families possess no catalytic kinase activity, they rely on the JAK family of tyrosine kinases to phosphorylate and activate downstream proteins involved in their signal transduction pathways. The receptors exist as paired polypeptides, thus exhibiting two intracellular signal-transducing domains.

[0067] JAKs associate with a proline-rich region in each intracellular domain that is adjacent to the cell membrane and called a boxl / box2 region. After the receptor associates with its respective cytokine / ligand, it goes through a conformational change, bringing the two JAKs close enough to phosphorylate each other. The JAK autophosphorylation induces a conformational change within itself, enabling it to transduce the intracellular signal by further phosphorylating and activating transcription factors called STATs (Signal Transducer and Activator of Transcription, or Signal Transduction And Transcription). The activated STATs dissociate from the receptor and form dimers before translocating to the cell nucleus, where they regulate transcription of selected genes.

[0068] Some examples of the molecules that use the JAK / STAT signaling pathway are colonystimulating factor, prolactin, growth hormone, and many cytokines. Janus Kinases have also been reported to have a role in the maintenance of X chromosome inactivation.

[0069] JAK inhibitors are used for the treatment of atopic dermatitis and rheumatoid arthritis. They are also being studied in psoriasis, polycythemia vera, alopecia, essential thrombocythemia, ulcerative colitis, myeloid metaplasia with myelofibrosis and vitiligo.

[0070] According to embodiments of the pharmaceutical composition, dosage form, combination, or kit according to the above description,

[0071] (a) the recombinant protein comprising

[0072] (i) interleukin-2 (IL2) and

[0073] (ii) a targeting entity that binds to fibronectin, and

[0074] (b) the Janus Kinase (JAK) inhibitor are administered or taken simultaneously or sequentially.

[0075] According to one embodiment the Janus Kinase (JAK) inhibitor is administered or taken before the recombinant protein comprising interleukin-2 (IL2) and a targeting entity is administered.

[0076] According to one embodiment the Janus Kinase (JAK) inhibitor is administered or taken after the recombinant protein comprising interleukin-2 (IL2) and a targeting entity is administered.

[0077] According to one embodiment the Janus Kinase (JAK) inhibitor is administered or taken simultaneously with the recombinant protein comprising interleukin-2 (IL2) and a targeting entity is administered.

[0078] According to one embodiment the Janus Kinase (JAK) inhibitor is administered orally, while the recombinant protein comprising interleukin-2 (IL2) and a targeting entity is administered i.v. or s.c. According to embodiments of the invention, the targeting entity binds to

[0079] • extra-domain A (ED-A) of fibronectin, or

[0080] • extra-domain B (ED-B) of fibronectin

[0081] Fibronectin is a high-molecular weight (-500—600 kDa) glycoprotein of the extracellular matrix that binds to membrane-spanning receptor proteins called integrins. Fibronectin also binds to other extracellular matrix proteins such as collagen, fibrin, and heparan sulphate proteoglycans (e.g., syndecans).

[0082] Fibronectin exists as a protein dimer, consisting of two nearly identical monomers linked by a pair of disulfide bonds. The fibronectin protein is produced from a single gene, but alternative splicing of its pre-mRNA leads to the creation of several isoforms.

[0083] Two types of fibronectin are present in vertebrates:

[0084] • soluble plasma fibronectin (formerly called "cold-insoluble globulin", or Cig) is a major protein component of blood plasma (300 pg / ml) and is produced in the liver by hepatocytes.

[0085] • insoluble cellular fibronectin is a major component of the extracellular matrix. It is secreted by various cells, primarily fibroblasts, as a soluble protein dimer and is then assembled into an insoluble matrix in a complex cell-mediated process.

[0086] Fibronectin plays a major role in cell adhesion, growth, migration, and differentiation, and it is important for processes such as wound healing and embryonic development. Altered fibronectin expression, degradation, and organization have been associated with a number of pathologies, including cancer, arthritis, and fibrosis.

[0087] Fibronectin isoform B-FN is one of the best-known markers of angiogenesis (see e.g., WO97 / 45544). An extra domain “ED-B” of 91 amino acids is found in the B-FN isoform and is identical in mouse, rat, rabbit, dog and man. B-FN 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. Fibronectin isoform A-FN (extra domain A, ED-A) is another cancer-related marker. ED-A is a known ligand for the a4pi integrin receptor, ED-A, which can also be inserted in the fibronectin transcript by a mechanism of alternative splicing, has been shown to preferentially accumulate around new blood vessels in certain tumors (see e.g., W02008 / 120101).

[0088] According to embodiments of the invention, the targeting entity that binds to fibronectin comprises an anti-fibronectin antibody, or a target binding fragment or derivative thereof.

[0089] According to embodiments of the invention, the JAK kinase inhibitor is specific for JAK1

[0090] As used herein, the term “specific for JAK1” is meant to refer to those JAK kinase inhibitors which bind to JAK1, and optionally to yet another JAK subtype.

[0091] As used herein, the term “specific for JAK3” is meant to refer to those JAK kinase inhibitors which bind to JAK3, and optionally to yet another JAK subtype.

[0092] According to embodiments of the invention, the JAK kinase inhibitor is Upadacitinib.

[0093] Upadacitinib, sold under the brand name Rinvoq®, is a medication used for the treatment of rheumatoid arthritis, psoriatic arthritis, atopic dermatitis, ulcerative colitis, Crohn's disease, ankylosing spondylitis, giant cell arteritis and non-radiographic axial spondyloarthritis.

[0094] Interestingly, Upadacitinib has a moderate potency (IC50 = 45 nM), compared to other JAK1 inhibitors like Abrocitinib (reported IC50 = 29 nM) andBaricitinib (IC50 = 5.9 nM). Still, surprisingly, Upadacitinib proved to have the strongest dampening efficacy on systemic toxicity of targeted IL2, while leaving the targeted IL2’s site-directed anticancer activity unaffected.

[0095] Table 1: Examples of JAK inhibitors

[0096] **potency determined as IC5o in cell-free assay, data taken from www.medchemexpress.com

[0097] The inventors of the present invention have surprisingly shown that JAK inhibitors, when coadministered (either simultaneously, or sequentially), with a targeted IL2, reduce the systemic toxicity thereof without affecting the anti-cancer activity of the targeted IL2. Even more surprisingly, this applies, in particular, to Upadacitinib, which, as shown in the above table, has a relatively moderate potency regarding the inhibition of JAK1, yet proved to have the strongest dampening efficacy on systemic toxicity of targeted IL2.

[0098] Even more surprisingly, it was shown that Upadacitinib (IC50 for JAK1 : 45 nM) proved to have a stronger dampening effect on the toxicity of targeted IL2, not only compared to other JAK1 inhibitors with higher potency, like Ruxolitinib (IC50 for JAK1 : 3.3 nM), but also compared to the JAK1 / JAK3 inhibitor Peficitinib (IC50 for JAK1 : 3.9 nM, IC50 for JAK3: 0.7 nM). .

[0099] Considering that both JAK1 and JAK3 mediate the IL2 signalling cascade, it would have been expected that the JAK1 / JAK3 Peficitinib inhibitor would prove to be superior to Upadacitinib, which only inhibits JAK1, and has, furthermore, a lower potency (see table 1). Accordingly, the combination with Peficitinib would have been expected to out-perform the combination with Upadacitib, which is a pure JAK1 inhibitor.

[0100] According to embodiments of the invention, the anti-fibronectin antibody, or a target binding fragment or derivative a) comprises a set of three heavy chain and three light chain complementarity determining regions (CDR) selected from

[0101] • SEQ ID NOs: 6, 7, 8, 9, 10, and 11

[0102] • SEQ ID NOs: 21, 22, 23, 24, 25, and 26, and / or b) comprises the heavy chain / light chain variable domain (HCVD / LCVD) pairs set forth in the following pairs of SEQ ID NOs:

[0103] • 1 and 3;

[0104] • 13 and 15, and / or c) comprises the heavy chain / light chain variable domains (HCVD / LCVD) pairs of b), with the proviso that

[0105] • the HCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and / or

[0106] • the LCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and / or d) the heavy chain / light chain variable domains (VD) pairs of b) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the respective SEQ ID NO, said antibody or fragment or derivative being capable to bind to fibronectin

[0107] SEQ ID NOs: 1 and 3 are the VH / VL sequences that are comprised, inter alia, in the anti-ED- B antibody LI 9. SEQ ID NOs: 6, 7, 8, 9, 10, and 11 are the CDR sequences that are comprised, inter alia, in the anti-ED-B antibody LI 9.

[0108] SEQ ID NOs: 13 and 15 are the VH / VL sequences that are comprised, inter alia, in the anti- ED-A antibody F8. SEQ ID NOs: 21, 22, 23, 24, 25, and 26 are the CDR sequences that are comprised, inter alia, in the anti-ED-A antibody F8. The anti-ED-B antibody L19 is described, inter alia, in W02003 / 076469. L19 adopts the format of a scFv comprising SEQ ID NO: 28. The anti-ED-A antibody F8 is described, inter alia, in W02008 / 120101. Being a diabody, F8 is a dimer of molecules each of which comprise SEQ ID NO: 29 [F8 VH-GGSGG(5aa)-F8 VL]2.

[0109] “Percentage of sequence identity” as used herein, is determined by comparing two optimally aligned biosequences (amino acid sequences or polynucleotide sequences) over a comparison window, wherein the portion of the corresponding sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence, which does not comprise additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0110] The terms “identical” or percent “identity,” in the context of two or more nucleic acids or polypeptide sequences, refer to two or more sequences or subsequences that are the same sequences. Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., at least 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity over a specified region, or, when not specified, over the entire sequence of a reference sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. The disclosure provides polypeptides that are substantially identical to the polypeptides exemplified herein. With respect to amino acid sequences, identity or substantial identity can exist over a region that is at least 5, 10, 15 or 20 amino acids in length, optionally at least about 25, 30, 35, 40, 50, 75 or 100 amino acids in length, optionally at least about 150, 200 or 250 amino acids in length, or over the full length of the reference sequence. With respect to shorter amino acid sequences, e.g., amino acid sequences of 20 or fewer amino acids, substantial identity exists when one or two amino acid residues are conservatively substituted, according to the conservative substitutions defined herein. A “variable domain” when used in reference to an antibody or a heavy or light chain thereof is intended to mean the portion of an antibody which confers antigen binding onto the molecule and which is not the constant region. The term is intended to include functional fragments thereof which maintain some or all of the binding function of the whole variable region. Variable region binding fragments include, for example, functional fragments such as Fab, F(ab)2, Fv, single chain Fv (scFv), diabodies and the like. Such functional fragments are well known to those skilled in the art. Accordingly, the use of these terms in describing functional fragments of a heteromeric variable region is intended to correspond to the definitions well known to those skilled in the art. Such terms are described in, for example, Huston et al., (1993) or Pliickthun and Skerra (1989).

[0111] 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 acids in length. Suitable linker sequences are known in the art.

[0112] In a diabody, a heavy chain variable domain (VH) is connected to a light chain variable domain (VL) on the same polypeptide chain. The VH and VL domains are connected by a peptide linker that is too short to allow pairing between the two domains (generally around 5 amino acids). This forces pairing with the complementary VH and VL domains of another chain.

[0113] According to embodiments, the HCVD and / or LCVD has a sequence identity of > 81 %; > 82 %; > 83 %; > 84 %; > 85 %; > 86 %; > 87 %; > 88 %; > 89 %; > 90 %; > 91 %; > 92 %; > 93 %; > 94 %; > 95 %; > 96 %; > 97 %; > 98 %; > 99 %; or 100 % to the respective SEQ ID NO.

[0114] According to one embodiment of the invention, at least one amino acid substitution is a conservative amino acid substitution.

[0115] A “conservative amino acid substitution”, as used herein, has a smaller effect on antibody function than a non-conservative substitution. Although there are many ways to classify amino acids, they are often sorted into six main groups on the basis of their structure and the general chemical characteristics of their R groups. In some embodiments, a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. For example, families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with

[0116] • basic side chains (e.g., lysine, arginine, histidine),

[0117] • acidic side chains (e.g., aspartic acid, glutamic acid),

[0118] • uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),

[0119] • nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan),

[0120] • beta-branched side chains (e.g., threonine, valine, isoleucine) and

[0121] • aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0122] Other conserved amino acid substitutions can also occur across amino acid side chain families, such as when substituting an asparagine for aspartic acid in order to modify the charge of a peptide. Conservative changes can further include substitution of chemically homologous nonnatural amino acids (i.e. a synthetic non-natural hydrophobic amino acid in place of leucine, a synthetic non-natural aromatic amino acid in place of tryptophan).

[0123] According to one embodiment of the invention, the antibody or fragment has a target binding affinity of > 50 % to fibronectin ED-A, or ED-B, respectively, compared to that of the antibody or fragment according to the above description.

[0124] As used herein the term “binding affinity” is intended to mean the strength of a binding interaction and therefore includes both the actual binding affinity as well as the apparent binding affinity. The actual binding affinity is a ratio of the association rate over the dissociation rate. Therefore, conferring or optimizing binding affinity includes altering either or both of these components to achieve the desired level of binding affinity. The apparent affinity can include, for example, the avidity of the interaction. For example, a bivalent heteromeric variable region binding fragment can exhibit altered or optimized binding affinity due to its valency. A suitable method for measuring the affinity of a binding agent is through surface plasmon resonance (SPR). This method is based on the phenomenon which occurs when surface plasmon waves are excited at a metal / liquid interface. Light is directed at, and reflected from, the side of the surface not in contact with sample, and SPR causes a reduction in the reflected light intensity at a specific combination of angle and wavelength. Biomolecular binding events cause changes in the refractive index at the surface layer, which are detected as changes in the SPR signal. The binding event can be either binding association or dissociation between a receptorligand pair. The changes in refractive index can be measured essentially instantaneously and therefore allow for determination of the individual components of an affinity constant. More specifically, the method enables accurate measurements of association rates (kon) and dissociation rates (koir).

[0125] Measurements of konand koir values can be advantageous because they can identify altered variable regions or optimized variable regions that are therapeutically more efficacious. For example, an altered variable region, or heteromeric binding fragment thereof, can be more efficacious because it has, for example, a higher konvalue compared to variable regions and heteromeric binding fragments that exhibit similar binding affinity. Increased efficacy is conferred because molecules with higher konvalues can specifically bind and inhibit their target at a faster rate. Similarly, a molecule of the invention can be more efficacious because it exhibits a lower koir value compared to molecules having similar binding affinity. Increased efficacy observed with molecules having lower koir rates can be observed because, once bound, the molecules are slower to dissociate from their target. Although described with reference to the altered variable regions and optimized variable regions of the invention including, heteromeric variable region binding fragments thereof, the methods described above for measuring association and dissociation rates are applicable to essentially any antibody or fragment or fragment thereof for identifying more effective binders for therapeutic or diagnostic purposes.

[0126] According to embodiments of the invention, the antibody or fragment or derivative that binds to fibronectin is provided in a single chain Fv (scFv) format, with the structure “VH-linker- VL”, and optionally

[0127] • the targeted IL2 is fused, directly or via a linker, to the C-terminus of the VL domain of the scFv.

[0128] According to embodiments of the invention, • the antibody or fragment or derivative that binds to fibronectin is provided in a single chain Fv format (scFv) or in a diabody format, preferably comprising the amino acid sequence of SEQ ID NO: 28 or 29

[0129] • the targeted IL2 comprises the amino acid sequence of SEQ ID NO: 5 or 20.

[0130] According to another aspect of the invention, the pharmaceutical composition, dosage form, combination, or kit according to the above description is provided for (the manufacture of a medicament consisting of at least one dosage form for) use in the treatment of a human or mammalian patient

[0131] (i) being diagnosed for,

[0132] (ii) suffering from or

[0133] (iii) being at risk of developing cancer.

[0134] This language is deemed to encompass both the “Swiss type” claim language accepted in some countries (in this case, brackets are deemed absent) and EPC2000 language (in this case, brackets and content within the brackets are deemed absent).

[0135] According to another aspect of the invention, a method for treating a human or mammalian patient is provided, which method comprises administration of one or more effective amounts of the pharmaceutical composition, dosage form combination or kit according to the above description, wherein the human or mammalian patient

[0136] (i) is diagnosed for,

[0137] (ii) suffers from or

[0138] (iii) is at risk of developing cancer.

[0139] According to embodiments, said cancer is at least one selected from the group consisting of solid or non-solid cancer, malignant lymphoma, liver cancer, lymphoma, leukaemia (e.g. acute myeloid leukaemia), sarcomas, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, head and neck cancer, oesophageal cancer, pancreatic cancer (e.g. pancreatic adenocarcinoma), renal cancer (e.g. clear cell renal adenocarcinoma), stomach cancer and / or cerebral cancer. EXAMPLES

[0140] While the invention has been illustrated and described in detail in the drawings and foregoing description, such illustration and description are to be considered illustrative or exemplary and not restrictive; the invention is not limited to the disclosed embodiments. Other variations to the disclosed embodiments can be understood and effected by those skilled in the art in practicing the claimed invention, from a study of the drawings, the disclosure, and the appended claims. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims should not be construed as limiting the scope.

[0141] All amino acid sequences disclosed herein are shown from N-terminus to C-terminus; all nucleic acid sequences disclosed herein are shown 5'->3'.

[0142] Example 1: Preparation of the combination partners

[0143] 1.1. Upadacitinib preparation

[0144] Lyophilized Upadacitinib was initially dissolved in 100% DMSO. For in vivo administration, Upadacitinib was prepared at a dose of 20 mg / kg in 5% DMSO + 5% Tween-20 in deionized water. Chemical structure of Upadacitinib is shown in Figure 8.

[0145] 1.2. Protein expression and characterization

[0146] The F8-IL2 immunocytokine used in the present experiments is a fusion protein consisting of the anti-EDA F8 antibody fragment, fused to murine IL2 at the N-terminus. The cloning and production of the protein have been previously described by De Luca et al (2017). The product was purified from the cell culture medium by affinity chromatography using a Protein A affinity column. After dialysis into PBS pH 7.4, the quality of the protein was assessed by SDS- PAGE and by Size-exclusion chromatography on a Superdex 200 Increase 10 / 300 GL column mounted on an AKTA FPLC. The L19-IL2 immunocytokine, consisting of the anti-EDB L19 antibody fragment, fused to the N-terminus of human IL2, was produced according to the protocol reported in WO2023 / 180409. The structures of F8-IL2 and of L19-IL2 are shown in Figure 9.

[0147] 1.3. Results

[0148] The proteins F8-IL2 (Figure 1A) and L19-IL2 (Figure IB) were pure and ran at the correct molecular weight.

[0149] Example 2: In vitro screening of inhibitors

[0150] In these experiments, the inhibitory effect of different JAK kinase inhibitors was measured on systemic T cell responses as evoked by administration of targeted IL2, including proliferation of T cells (Figure 2), IFNy-release (Figure 3) and TNFa release (Figure 4).

[0151] 2.1. Quantification of proliferation and cytokine release

[0152] CTLL2 (murine cytotoxic T lymphocyte cell line 2) cells were analyzed for their ability to release cytokines and proliferate upon incubation with different small-molecule inhibitors. Baricitinib (HY-15315), Tofacitinib (HY-40354), Upadacitinib (HY-19569), Abrocitinib (HY- 107429), Ruxolitinib (HY-50856) and Peficitinib (HY-19568) were purchased from MedChemExpress LLC. CTLL2 cells were initially starved for 24 hours in RPMI medium supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were then resuspended at a density of 0.25 x 106cells / mL in complete RPMI, and 100 pL of the cell suspension was incubated with increasing concentrations of the inhibitors in the presence of F8-IL2 (InM) or L19-IL2 (InM). After 72 hours, supernatants were collected and the concentration of IFN-y and TNF-a was measured using the ELISA MAXTM Set Mouse IFN-y kit and TNF-a kit (BioLegend). Cell proliferation was assessed on the pellets with Cell Titer Aqueous One Solution (Promega).

[0153] 2.2. Comparative study to measure potency of Upadacitinib with F8-IL2 or L19-IL2 CTLL2 cells were incubated with 1 nM of F8-IL2 or L19-IL2 in combination with increasing doses of Upadacitinib. After 72h, cell proliferation was measured using the CellTiter96® AQueous One Solution Cell Proliferation Assay.

[0154] 2.3. Results

[0155] Figures 2-4 show that the JAK inhibitors have a dampening effect on systemic toxicity of targeted IL2 (targeting ED-A, as exemplified by F8-IL2 or targeting ED-B as exemplified by L19-IL2), with Upadacitinib showing the strongest effects despite its relatively smallest potency when JAK inhibition alone is considered (IC50 = 45 nM).

[0156] Surprisingly, Abrocitinib, the JAK1 inhibitor with potent IC50 reported in the literature (29 nM), showed less efficacy in modulating IL2 activity compared to Upadacitinib.

[0157] Even more surprisingly, Figure 10 shows that the JAK1 / JAK3 inhibitor Peficitinib showed less efficacy in modulating IL2 activity compared to the JAK1 inhibitor Upadacitinib.

[0158] Moreover, Figure 5 shows that Upadacitinib elicits the same potency in dampening systemic T cell responses (= T cell proliferation) of both F8-IL2 (targeting ED-A), and L19-IL2 (targeting ED-B). As a consequence, the results shown in Figures 2-4 can be extrapolated also to a combination of the JAK inhibitors with targeted IL2, targeting ED-B, as exemplified by LI 9- IL2).

[0159] Example 3: Therapy experiments

[0160] 3.1. Preparation of the tumor cells

[0161] MC-38 (murine colon adenocarcinoma) cells, derived from a C57BL / 6 mouse were cultured in DMEM medium supplemented with Fetal Bovine Serum (10%) and antibiotic-antimycotic (1%) following the supplier's protocol and kept in culture for no longer than 10 passages with a confluence lower than 90%. Eight-week-old female C57BL / 6 mice were used for the experiment.

[0162] 3.2. Tumor implantation MC-38 cells were grown to 80% confluence and detached with Trypsin-EDTA 0.05%. Cells were washed, counted and re-suspended in HBSS to a final concentration of 1 x 107cells ml-1. Aliquots of 1 x 106cells (100 pl of the suspension) were injected subcutaneously in the right flank of each animal. Tumor volume was determined with the following formula: (length x width2x 0.5) measured with a caliper.

[0163] 3.3. Therapy experiments

[0164] When tumors reached an average of about 100 mm3, MC-38 tumor-bearing mice were randomized, and different treatments were started. 4 different groups (4 / 5 mice per group) received the following treatments:

[0165] (i) i.v. vehicle (saline solution) on day 6, 8 and 10 after tumor implantation

[0166] (ii) s.c. Upadacitinib (20 mg / kg, deionized water + 5% DMSO + 5% Tween) on day 6, 8 and 10 after tumor implantation

[0167] (iii) i.v. F8-IL2 (100 pg, saline solution) on day 6, 8 and 10 after tumor implantation

[0168] (iv) s.c Upadacitinib (20 mg / kg, deionized water + 5% DMSO + 5% Tween) and i.v. F8-IL2 (100 pg, saline solution) on day 6, 8 and 10 after tumor implantation.

[0169] In group (iv) Upadacitinib was administered 20 minutes before and 6 hours after the administration of F8-IL2.

[0170] Efficacy of the different treatments was monitored by daily tumor volume measurement.

[0171] Toxicity of the different treatments was monitored by daily body weighing and general appearance of the animals. Twenty-four hours after last injection mice were euthanized and lungs and livers were weighted to further assess post mortem toxicity.

[0172] For quantification of IFNy levels in the plasma 24h after a full therapy cycle, mice were euthanized, blood was transferred to heparin tubes and plasma was collected after centrifugation at 1500xg for 15 minutes. IFNy levels were determined using the ELISA MAX™ Set Murine IFNy kit. 3.4. Results

[0173] 3.4.1. Therapy studies

[0174] The addition of Upadacitinib to high doses of a targeted-IL2 has maintained the significant anticancer activity induced by the immunocytokine (Figure 6A).

[0175] 3.4.2. Toxicity

[0176] As expected, the administration of high doses of a targeted-IL2 (5mg / kg) caused a significant weight loss in the treated mice. The toxicity effect was more evident after the second injection, between days 10-12.

[0177] However, when a targeted-IL2 was combined with Upadacitinib (20mg / kg), the weight loss was negligible and comparable to the non-treated group (Figure 6B).

[0178] Therefore, the combination of a targeted-IL2 with Upadacitinib significantly reduces the toxicity of IL2, without affecting the anti -tumor activity of targeted IL2.

[0179] Prevention of toxicity was confirmed by the weights of the lungs (Figure 6C) and of the livers (Figure 6D) which were comparable to the vehicle group for the Upadacitinib and the combination groups, but significantly higher for the targeted-IL2 group indicating lung and liver edema. As a further confirmation, plasma levels of fFNy (a marker of inflammation) were significantly lower in the combination group as compared to the targeted-IL2 group (Figure 6E).

[0180] Example 4: Comparative study to measure potency of Upadacitinib and Sunitinib with F8-IL2 or L19-IL2

[0181] CTLL2 cells were incubated with InM of F8-IL2 or L19-IL2 in combination with increasing doses of Upadacitinib or Sunitinib (HY- 10255 A, MedChemExpress LLC). After 72h, cell proliferation was measured using the CellTiter96® AQueous One Solution Cell Proliferation Assay. Sunitinib is a small-molecule, multi -targeted receptor tyrosine kinase (RTK) inhibitor that inhibits tyrosine kinase activity of all receptors for platelet-derived growth factor (PDGF-Rs) and vascular endothelial growth factor receptors (VEGFRs). Sunitinib also inhibits CD117 (c- KIT), the receptor tyrosine kinase that (when improperly activated by mutation) drives the majority of gastrointestinal stromal cell tumors.

[0182] As can be seen, the dampening effect of the JAK inhibitor Upadacitinib on systemic toxicity of targeted IL2 (targeting ED-A, as exemplified by F8-IL2, Figure 7A, or targeting ED-B, as exemplified by L19-IL2 Figure 7B), was higher than that of the tyrosine kinase inhibitor Sunitinib.

[0183] Example 5: Comparative study to measure potency of Upadacitinib and Peficitinib with F8-IL2 or L19-IL2

[0184] A similar experiment as in example 4 was carried out, with the difference that a combination of Peficitinib with F8-IL2 or L19-IL2 was tested against the respective combination of Upadacitinib with F8-IL2 or L19-IL2. As can be seen, the dampening effect of the JAK inhibitor Upadacitinib on systemic toxicity of targeted IL2 (targeting ED-A, as exemplified by F8-IL2, Figure 10A, or targeting ED-B, as exemplified by L19-IL2 Figure 10B), was higher than that of the JAK1 / JAK3 inhibitor Peficitinib.

[0185] This is surprising not only because Upadacitinib has a significantly lower potency regarding the inhibition of JAK1, but also because IL2 signalling activates both JAK1 and JAK3, so that one would have expected better efficacy of the JAK1 / JAK3 inhibitor Peficitinib relative to Upadacitinib, which only inhibits JAKE

[0186] References

[0187] • Neri & Bicknell (2005) Nat Rev Cancer, 5, 436

[0188] • Pasche et al. (2011) J Biotechnology, 154, 84-92

[0189] • Kaspar et al. (2007) Cancer Res, 67, 4940-4948

[0190] • Rosenberg (2014) J Immunol, 192, 5451-5458

[0191] • Dutcher et al (2014) J Immunother Cancer, 2, 26

[0192] • Yang et al. (2003) J Clin Oncol, 21, 3127-3132 • Atkins et al. (2000) Cancer J Sci Am, 6, S11-S14

[0193] • Fyfe et al. (1995) Clin Oncol, 13, 688-696

[0194] • Kammula et al. (1998) Cancer, 83, 797-805

[0195] • Dutcher et al. (1997) Cancer J Sci Am, 3, S73-S78

[0196] • Wagner et al (2008) Clin Cancer Res 14, 4951

[0197] • Schliemann et al (2009 Blood 113, 2275

[0198] • Zegers et al (2015) Clin Cancer Res 21, 1151

[0199] • Frey et al., (2010) J Urol, 184, 2540

[0200] • Gutbrodt et al., (2013) Sci Transl Med, 5

[0201] • Wieckowski et al., (2015) Lung Cancer, 9-15

[0202] • Hutchmaker et al (2019) Cancer Immunol Res 7, 572

[0203] • Ziffels et al., (2018) Immunotherapy, 10, 177

[0204] • Pretto et al (2014) Cancer Immunol Immunother. 2014 Sep;63(9):901-10.

[0205] • Moschetta et al., (2012) Cancer Res 72, 1814

[0206] • Cazzamalli et al., (2018) Clin Cancer Res, 24, 3656

[0207] • Huston et al., (1993) Int Rev Immunol 10, 195

[0208] • Pliickthun and Skerra, Meth. Enzymol., 178:497-515 (1989)

[0209] • Villa et al, Int J Cancer. 2008 Jun

[0210] • De Luca et al. Mol Cancer Ther (2017), 16:2442-51.

[0211] • WO97 / 45544

[0212] • WO2011 / 015333

[0213] • WO1999 / 058570

[0214] • W02003 / 076469

[0215] • W02005 / 023318

[0216] • W02001 / 062298

[0217] • W02007 / 115837

[0218] • W02009 / 089858

[0219] • WO2013 / 010749

[0220] • WO2013 / 045125

[0221] • WO2018 / 115377

[0222] • WO2018 / 154517

[0223] • W02020 / 070150

[0224] • W02008 / 120101 1 • WO2018 / 069467

[0225] • WO2007 / 128563

[0226] • W02009 / 056268

[0227] • W02006 / 119897

[0228] • W02013 / 014149

[0229] • WO2019 / 154986

[0230] • WO2023 / 131611

[0231] • WO2018 / 224550

[0232] • WO2017 / 009469

[0233] • WO2022 / 018126

[0234] • WO2022 / 214664

[0235] • WO97 / 45544

[0236] • W02003 / 076469

[0237] • WO2023 / 180409

[0238] Sequences

[0239] The following sequences form part of the disclosure of the present application. A WIPO ST 26 compatible electronic sequence listing is provided with this application, too. For the avoidance of doubt, if discrepancies exist between the sequences in the following table and the electronic sequence listing, the sequences in this table shall be deemed to be the correct ones.

[0240] In some cases, signal peptides may be encompassed in the reproduced sequences. In such case, the sequences shall be deemed disclosed with and without signal peptides. A readily available tool to identify signal peptides in a given protein sequence is SignalP - 6.0 provided by Dansk Technical University under https: / / services.healthtech.dtu.dk / service.php7SignalP. The same applies to His tags or C-Myc tags, if existing. The skilled artisan is able to identify such tags and understand that such tags do not necessarily make it into the product that is actually used.

[0241] Table 2: Sequence listing

Claims

What is claimed is:

1. A pharmaceutical composition comprising(a) a recombinant protein comprising(i) interleukin-2 (IL2) and(ii) a targeting entity which binds to fibronectin, which targeting entity comprises an antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin, or a target binding fragment thereof, and(b) a Janus Kinase (JAK) inhibitor, wherein the JAK Kinase inhibitor is Upadacitinib.

2. A dosage form comprising(a) a recombinant protein comprising(i) interleukin-2 (IL2) and(ii) a targeting entity which binds to fibronectin, which targeting entity comprises an antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin, or a target binding fragment thereof, and(b) a Janus Kinase (JAK) inhibitor, in a pharmaceutically acceptable carrier, wherein the JAK Kinase inhibitor is Upadacitinib,3. A combination comprising at least(a) a recombinant protein comprising(i) interleukin-2 (IL2) and(ii) a targeting entity which binds to fibronectin, which targeting entity comprises an antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin, or a target binding fragment thereof, and(b) a Janus Kinase (JAK) inhibitor, wherein the JAK Kinase inhibitor is Upadacitinib.

4. A kit of dosage forms, comprising at least(a) a first dosage form comprises a recombinant protein comprising(i) interleukin-2 (IL2) and(ii) a targeting entity which binds to fibronectin, which targeting entity comprises an antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin, or a target binding fragment thereof, in a pharmaceutically acceptable carrier, and(b) a second dosage form comprises a Janus Kinase (JAK) inhibitor in a pharmaceutically acceptable carrier, wherein the JAK Kinase inhibitor is Upadacitinib.

5. The pharmaceutical composition, dosage form, composition or kit according to any one of claims 1 - 4, wherein the antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin a) comprises a set of three heavy chain and three light chain complementarity determining regions (CDR) selected from• SEQ ID NOs: 6, 7, 8, 9, 10, and 11• SEQ ID NOs: 21, 22, 23, 24, 25, and 26, and / or b) comprises the heavy chain / light chain variable domain (HCVD / LCVD) pairs set forth in the following pairs of SEQ ID NOs:• 1 and 3;• 13 and 15.

6. The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 1 - 5, wherein• the antibody or fragment that binds to fibronectin is provided in a single chain Fv format (scFv) or in a diabody format, preferably comprising the amino acid sequence of SEQ ID NO: 28 or 29• the targeted IL2 comprises the amino acid sequence of SEQ ID NO: 5 or 20.

7. A pharmaceutical composition, dosage form, combination or kit of dosage forms comprising(a) a recombinant protein comprising(i) interleukin-2 (IL2) and(ii) a targeting entity that binds to fibronectin, and(b) a Janus Kinase (JAK) inhibitorwherein optionally the recombinant protein and the Janus Kinase (JAK) inhibitor are provided in a separate dosage form, and wherein optionally the recombinant protein and / or the Janus Kinase (JAK) inhibitor are provided in, or together with, a pharmaceutically acceptable carrier.

8. The pharmaceutical composition, dosage form, combination, or kit according to claim 7, wherein the targeting entity binds to• extra-domain A (ED-A) of fibronectin, or• extra-domain B (ED-B) of fibronectin.

9. The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 7 - 8, wherein the targeting entity that binds to fibronectin comprises an anti-fibronectin antibody, or a target binding fragment thereof.

10. The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 7 - 9, wherein the JAK kinase inhibitor is specific for JAKE11. The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 7 - 10, wherein the JAK kinase inhibitor is Upadacitinib.

12. The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 7 - 11, wherein the anti-fibronectin antibody, or fragment a) comprises a set of three heavy chain and three light chain complementarity determining regions (CDR) selected from• SEQ ID NOs: 6, 7, 8, 9, 10, and 11• SEQ ID NOs: 21, 22, 23, 24, 25, and 26, and / or b) comprises the heavy chain / light chain variable domain (HCVD / LCVD) pairs set forth in the following pairs of SEQ ID NOs:1 and 3;13 and 15, and / orc) comprises the heavy chain / light chain variable domains (HCVD / LCVD) pairs of b), with the proviso that• the HCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and / or• the LCVD has a sequence identity of > 80 % to the respective SEQ ID NO, and / or d) the heavy chain / light chain variable domains (VD) pairs of b) or b), with the proviso that at least one of the HCVD or LCVD has up to 10 amino acid substitutions relative to the respective SEQ ID NO, said antibody or fragment being capable to bind to fibronectin.

13. The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 7 - 12, wherein• the antibody or fragment that binds to fibronectin is provided in a single chain Fv format (scFv) or in a diabody format, preferably comprising the amino acid sequence of SEQ ID NO: 28 or 29• the targeted IL2 comprises the amino acid sequence of SEQ ID NO: 5 or 20.

14. The pharmaceutical composition, dosage form, combination, or kit according to any one of the aforementioned claims, wherein(a) the recombinant protein comprising(i) interleukin-2 (IL2) and(ii) the targeting entity that binds to fibronectin, and(b) the Janus Kinase inhibitor are administered or taken simultaneously or sequentially15. The combination, dosage form, composition or kit according to any one of the aforementioned claims, wherein the recombinant protein is an immunocytokine.

16. The combination, dosage form, composition or kit according to any one of the aforementioned claims, wherein, the recombinant protein is a fusion protein comprising interleukin-2 (IL2) and the antibody binding the extra-domain B (ED-B) or the extra-domain A (ED-A) of fibronectin17. The pharmaceutical composition, dosage form, combination, or kit according to any one of the aforementioned claims for (the manufacture of a medicament consisting of at least one dosage form for) use in the treatment in a human or mammalian patient(i) being diagnosed for,(ii) suffering from or(iii) being at risk of developing cancer.

18. A method for treating a human or mammalian patient, which method comprises administration of one or more effective amounts of the pharmaceutical composition, dosage form combination or Kit according to any one of the aforementioned claims, wherein the human or mammalian patient(i) is diagnosed for,(ii) suffers from or(iii) is at risk of developing cancer.

19. The use or method of any one of claims 17 and 18, wherein the cancer is at least one selected from the group consisting of solid or non-solid cancer, malignant lymphoma, liver cancer, lymphoma, leukaemia (e.g. acute myeloid leukaemia), sarcomas, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, head and neck cancer, oesophageal cancer, pancreatic cancer, pancreatic adenocarcinoma, renal cancer, clear cell renal adenocarcinoma, stomach cancer and / or cerebral cancer.

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