Combination of immunocytokines, including IL-12, and kinase inhibitors

Abstract

The present invention relates to a combination comprising (i) a recombinant protein comprising an antibody that binds to interleukin-12 (IL-12) and the extra domain B (ED-B) of fibronectin, or a target-binding fragment or derivative thereof, and (ii) a kinase inhibitor, and its use for the treatment of cancer (Figure 1).

Description

[Technical field]

[0001] The present invention relates to the field of immunoconjugates.

[0002] Incorporation by Reference All publications, patents, patent applications, and other documents cited in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application, or other document was individually indicated to be incorporated by reference. In the event of a conflict between the teachings of one or more documents incorporated herein and the present disclosure, the teachings of the present specification are intended to control. [Background technology]

[0003] Cytokines are important mediators of innate and adaptive immunity. Although many cytokines are used for therapeutic purposes in patients with advanced cancer, their administration is generally accompanied by severe toxicity, hindering their dose escalation into 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 to focus immune system stimulating activity at the site of disease while sparing normal tissues (Neri & Bicknell, 2005). However, genetic fusion of a cytokine to an antibody or antibody fragment to create an "immunocytokine" does not necessarily result in an immunocytokine that retains the tumor-targeting ability of the antibody. For example, the tumor targeting ability of certain GM-CSF fusions (Kaspar et al. (2007) Cancer Res, 67, 4940-4948) was found to be dose-dependent, whereas tumor targeting was completely abolished by certain interleukin-7 fusions (Pasche et al. (2011) J Biotechnology, 154, 84-92).

[0004] IL-12 is produced by antigen-presenting cells, such as macrophages and CDlc+ dendritic cells, and is expressed by natural killer (NK) cells, CD8 +Cytotoxic T cells, CD4 + It acts on T helper cells. Originally called natural killer cell stimulating factor, IL12 stimulates NK cells and CD8 + Promotes the cytotoxic activity of T cells and CD4 + Promotes polarization of T cells towards the type 1 phenotype.

[0005] Interestingly, human CD4 introduced into the xenogeneic environment of IL-12-free humanized mice + and CD8 + T cells preferentially differentiate into type 2 (IL4+ GATA3+) or mixed type 1 and 2 (IFNG+ TBET+ IL4+ GATA3+) subsets. Injection of mice with recombinant human IL-12 restored type 1 differentiation and improved cytotoxic immunity to viral challenge. In humans, genetic mutations in IL-12p40 and IL-12RB1, a component of the IL-12 receptor, have been observed in patients with recurrent mycobacteriosis, suggesting insufficient type 1 cell-mediated immunity. In mice, genetic deletion of IL-12RB2, another component of the IL-12 receptor, increases susceptibility to spontaneous autoimmunity, B cell malignancies, and lung cancer.

[0006] As a single agent, intravenous injection of recombinant IL-12 has shown modest clinical activity in a handful of patients with advanced melanoma and renal cell carcinoma, but interest in systemic administration of IL-12 has been limited because of deaths due to Clostridium perfringens sepsis in the first phase I trial.

[0007] In combination therapy, IL-12 has been used as an adjuvant to enhance cytotoxic immunity using melanoma antigen vaccines and peptide-pulsed peripheral blood mononuclear cells, and to promote NK cell-mediated killing of HER2-positive breast cancer cells in patients treated with trastuzumab.

[0008] Like many other cytokines, administration of recombinant human IL-12 is associated with severe toxicity, hindering its development as an anticancer agent.

[0009] Clinical trials in cancer patients have demonstrated promising therapeutic activity, but have also shown that recombinant human IL-12 is highly toxic to humans, with a maximum tolerated dose of 0.5 μg / kg of body weight.

[0010] Toxins, especially cytokines such as IL-12, have toxic side effects and therefore are difficult to administer in effective amounts and achieve high concentrations at the tumor site.

[0011] Previously, researchers have attempted to overcome these shortcomings by targeting the delivery of the IL-12 cytokine to the tumor environment, for example by conjugation with antibodies specific for antigens associated with cancer growth. These cytokine-antibody conjugates are often referred to as "immunocytokines."

[0012] The applicant has shown that the therapeutic index of the IL-12 cytokine can be improved by conjugating it to an antibody fragment, such as an scFv antibody fragment (WO2006 / 119897) or a single chain diabody (WO2013 / 014149).

[0013] Other versions of targeted IL-12, such as L19-IL12 with improved linkers (WO2019 / 122025, WO2021 / 209452), have also been reported by the applicant.

[0014] However, there remains a need to improve the therapeutic index of targeted IL-12. For example, it would be highly advantageous to find ways to reduce the toxicity associated with IL-12 while maintaining its anticancer activity.

[0015] To overcome the drawbacks associated with IL-12 therapy, it has been proposed to deliver IL-12 to the tumor site by antibodies against tumor-associated markers, thus increasing the local concentration of IL-12 at the tumor site and reducing the toxicity associated with systemic administration of IL-12. In particular, cytokine concentration at the tumor vascular level is an attractive therapeutic strategy because tumor neovasculature is more accessible to intravenously administered therapeutics than tumor cells, which helps to circumvent the problems associated with interstitial hypertension in solid tumors. Furthermore, angiogenesis is characteristic of most aggressive solid tumors. Angiogenesis is the growth of new blood vessels from pre-existing vessels. Tumors induce angiogenesis by secreting various growth factors (e.g., vascular endothelial growth factor). Tumor angiogenesis allows tumors to grow to more than a few millimeters in diameter and is also a prerequisite for tumor metastasis. The neovasculature formed as a result of angiogenesis forms the tumor neovasculature or tumor metastasis. Targeting IL-12 to the neovasculature should enable immunotherapy of a variety of different types of tumors.

[0016] The alternatively spliced ​​extra domain B (ED-B) of fibronectin is one of the best-characterized markers of angiogenesis and has been reported to be expressed around neovasculature and in the stroma of virtually all types of malignant solid tumors. Furthermore, even non-solid cancers such as leukemia may be treatable by targeting antigens on neovasculature. WO2011 / 015333 describes a method for the treatment of leukemia, including acute myeloid leukemia, by targeting bone marrow neovasculature.

[0017] The human monoclonal antibody L19, specific for this target, has been extensively described (WO1999 / 058570, WO2003 / 076469, WO2005 / 023318).

[0018] In addition, L19-based immunocytokines are currently being studied in phase I, II, and III clinical trials in cancer patients, including several cytokines, including IL-12. Summary of the Invention [Problem to be solved by the invention]

[0019] One object of the present invention is to broaden the range of therapeutic applications of the above identified immunocytokines.

[0020] Another object of the present invention is to provide new treatments for pathologies for which no suitable treatment currently exists.

[0021] Another object of the present invention is to improve the efficacy of treatment with the above immunocytokines. [Brief description of the drawings]

[0022] [Figure 1] SDS-PAGE analysis of purified L19-IL12 under non-reducing (NR) and reducing (R) conditions (left) and size-exclusion chromatography profile of L19-IL12 (right). [Figure 2A] Antitumor activity in tumor-bearing mice treated with three different doses of (i) vehicle, (ii) ruxolitinib, (iii) L19-IL12 alone or (iv) in combination with ruxolitinib. [Figure 2B] Toxicity of (i) vehicle, (ii) ruxolitinib 75 mg / kg, (iii) L19-IL12 (0.6 mg / kg) administered alone or (iv) in combination with ruxolitinib was expressed as change in body weight. [Figure 2C] Toxicity of (i) vehicle, (ii) ruxolitinib, (iii) L19-IL12 (0.9 mg / kg) administered alone or (iv) in combination with ruxolitinib was expressed as change in body weight. [Figure 2D] Toxicity of (i) vehicle, (ii) ruxolitinib, (iii) L19-IL12 (1.2 mg / kg) alone or (iv) in combination with ruxolitinib was expressed as body weight change. Days of treatment are indicated by black arrows. [Diagram 3] Diagram of the chemical structure of ruxolitinib. [Figure 4]An exemplary immunocytokine format using IL-12 and an anti-fibronectin antibody. [Diagram 5] In vitro quantification of interferon-γ levels in human cells incubated with L19-IL12 and different doses of JAK kinase inhibitors and dexamethasone. [Figure 6A] (i) Antitumor activity in tumor-bearing mice of vehicle, (ii) ruxolitinib, (iii) two high doses (2.4 mg / kg or 3.0 mg / kg) of L19-IL12 administered alone or in combination with ruxolitinib. [Figure 6B] Toxicity of (i) vehicle, (ii) ruxolitinib, (iii) L19-IL12 (2.4 mg / kg or 3.0 mg / kg) alone or (iv) in combination with ruxolitinib was expressed as body weight change. Days of treatment are indicated by black arrows. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The present invention provides, inter alia, combinations of immunocytokines, including IL-12, with kinase inhibitors. According to a first aspect of the present invention, (a)(i) interleukin 12 (IL-12) and (ii) Targeting Entity a targeted immunocytokine comprising (b) Kinase inhibitor A pharmaceutical composition is provided comprising: The present inventors unexpectedly recognized that kinase inhibitors reduce the toxicity of targeted IL-12 while leaving its anti-cancer activity unchanged.

[0024] In particular, the inventors found that when targeted immunocytokines, including interleukin-12 (IL-12), were administered to tumor-bearing mice in combination with such kinase inhibitors, the targeted IL-12 maintained its anti-cancer activity while at the same time exhibiting significantly reduced toxicity.

[0025] There was no a priori reason to expect that combination with a kinase inhibitor would reduce the toxicity of IL-12, but as the experimental results show, the results were even more surprising.

[0026] As used herein, the term "kinase inhibitor" (TKI) refers to compounds, mainly small molecules, that inhibit tyrosine kinases. Tyrosine kinases are enzymes responsible for the activation of many proteins through signal transduction cascades. Proteins are activated by the addition of phosphate groups to proteins (phosphorylation), a step that TKIs inhibit. TKIs are commonly used as anticancer drugs. TKIs act by four different mechanisms: they can compete with adenosine triphosphate (ATP), a substrate, or both, act allosterically, i.e. bind to sites other than the active site, and affect activity by conformational changes. Recently, TKIs have been shown to deprive tyrosine kinases of access to the Cdc37-Hsp90 molecular chaperone system on which they depend for cellular stability, leading to ubiquitination and degradation. Signal transduction therapy can also be used in proliferative and inflammatory diseases other than cancer.

[0027] As used herein, the term "immunocytokine" refers to a fusion protein consisting of a cytokine moiety fused to a targeting entity. Immunocytokine preparations specific for tumor-associated antigens on cell membranes have the potential to bridge tumor cells with specific leukocytes (e.g., T cells and NK cells), similar to what can be achieved by using bispecific antibodies. In contrast, immunocytokines targeting tumor-associated extracellular matrix components (e.g., splice isoforms of fibronectin and tenascin-C) are believed to exert their biological activity primarily through the dense fixation of the cytokine moiety to disease sites.

[0028] As used herein, the term "targeting entity" relates to a molecule capable of binding to a given target with high specificity and affinity. Such a targeting entity is, for example, an antibody or a fragment or derivative thereof.

[0029] Interleukin 12 (IL-12) is an interleukin naturally produced by dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigenic stimulation. IL-12 is composed of a bundle of four α-helices. It is a heterodimeric cytokine encoded by two genes, IL-12A (p35) and IL-12B (p40). The active heterodimer (termed "p70") and the homodimer of p40 are formed after protein synthesis.

[0030] IL-12 is involved in the differentiation of naive T cells into Th1 cells. It is known as a T cell stimulatory factor and can stimulate the growth and function of T cells. It stimulates the production of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α) from T cells and natural killer (NK) cells and suppresses the suppression of IFN-γ by IL-4. IL-12-producing T cells have a coreceptor called CD30 that is associated with the activity of IL-12.

[0031] IL-12 plays an important role in the activity of natural killer cells and T lymphocytes. IL-12 enhances the cytotoxic activity of NK cells and CD8+ cytotoxic T lymphocytes. There also appears to be a link between IL-2 and IL-12 signaling in NK cells. IL-2 stimulates the expression of two IL-12 receptors, IL-12R-β1 and IL-12R-β2, and maintains the expression of key proteins involved in IL-12 signaling in NK cells. Enhanced functional responses are indicated by IFN-γ production and target cell killing.

[0032] IL-12 also has antiangiogenic properties, and can inhibit the formation of new blood vessels. It does this by increasing the production of interferon gamma, which in turn increases the production of a chemokine called inducible protein-10 (IP-10 or CXCL10). IP-10 mediates this antiangiogenic effect. Because of IL-12's ability to induce an immune response and its antiangiogenic properties, there has been interest in testing it as an anticancer drug. However, no substantial activity has been observed in the tumors tested to date. There are links between IL-12 and psoriasis and inflammatory bowel disease that may be useful for treatment.

[0033] In some embodiments, the first subunit of the IL-12 protein is p40 and the second subunit is p35.

[0034] In some embodiments, the first subunit of the IL-12 protein is p40 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:1, or a fragment thereof, wherein the IL-12 protein is capable of activating the IL-12 receptor.

[0035] In some embodiments, the second subunit of the IL-12 protein is p35 comprising an amino acid sequence having at least 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence set forth in SEQ ID NO:3, or a fragment thereof, wherein the IL-12 protein is capable of activating the IL-12 receptor.

[0036] According to another aspect of the present invention, (a)(i) interleukin 12 (IL-12) and (ii) Targeting Entity A recombinant protein comprising (b) Kinase inhibitor in a pharma- ceutically acceptable carrier.

[0037] According to another aspect of the present invention, at least (a)(i) interleukin 12 (IL-12) and (ii) Targeting Entity A recombinant protein comprising (b) Kinase inhibitor A combination comprising:

[0038] According to another aspect of the present invention, at least (a)(i) interleukin 12 (IL-12) and (ii) Targeting Entity A first dosage form comprising a recombinant protein comprising the compound in a pharma- ceutically acceptable carrier; and (b) a second dosage form comprising a kinase inhibitor in a pharma- ceutically acceptable carrier. A dosage form kit comprising:

[0039] According to one embodiment of the pharmaceutical composition, dosage form, combination or kit according to the above description, (a)(i) interleukin 12 (IL-12) and (ii) Targeting Entity A recombinant protein comprising: (b) Kinase inhibitor are administered or taken simultaneously.

[0040] According to one embodiment of the pharmaceutical composition, dosage form, combination or kit according to the above description, (a)(i) interleukin 12 (IL-12) and (ii) Targeting Entity A recombinant protein comprising: (b) Kinase inhibitor are administered or taken sequentially.

[0041] According to one embodiment, a kinase inhibitor, such as a JAK kinase inhibitor, preferably ruxolitinib, is administered or taken prior to the recombinant protein comprising interleukin-12 (IL-12) and a targeting entity.

[0042] According to one embodiment of the pharmaceutical composition, dosage form, combination or kit according to the above description, the targeting entity comprises an anti-fibronectin antibody, or a target-binding fragment or derivative thereof.

[0043] According to one embodiment of the pharmaceutical composition, dosage form, combination or kit according to the above description, the targeting entity comprises an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment or derivative thereof.

[0044] Fibronectin is a high molecular weight (~500-600 kDa) glycoprotein of the extracellular matrix that binds to transmembrane receptor proteins called integrins and also to other extracellular matrix proteins such as collagen, fibrin, and heparan sulfate proteoglycans (e.g., syndecans).

[0045] 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, although several isoforms are generated by alternative splicing of its pre-mRNA.

[0046] There are two types of fibronectin in vertebrates: Soluble plasma fibronectin (previously called "cold-insoluble globulin", or CIg) is the major protein component of plasma (300 μg / ml) and is produced in the liver by hepatocytes. Insoluble cellular fibronectin is a major component of the extracellular matrix. It is secreted by a variety of cells, mainly fibroblasts, as a soluble protein dimer and then assembled into the insoluble matrix in a complex cell-mediated process.

[0047] Fibronectin plays a key role in cell adhesion, growth, migration and differentiation, and is important in processes such as wound healing and embryonic development. Alterations in fibronectin expression, degradation and organization are associated with many pathological conditions, including cancer, arthritis and fibrosis.

[0048] The fibronectin isoform B-FN is one of the best-known markers of angiogenesis (see, for example, WO1997 / 045544). B-FN isoforms contain an extra domain of 91 amino acids, "ED-B", and are identical in mouse, rat, rabbit, dog and human. B-FN accumulates around neovascular structures in progressive tumors and other tissues undergoing angiogenesis, such as the proliferative endometrium and some pathological ocular structures, but is not detectable in normal adult tissues.

[0049] The extra domain B (ED-B) of fibronectin is an attractive target for anti-cancer therapy, including the use of immunocytokines as discussed herein, particularly immunocytokines including the antibody L19 as discussed herein.

[0050] L19 is a human monoclonal scFv specific for the ED-B domain of fibronectin and has been previously described (WO1999 / 058570; WO2003 / 076469, WO2005 / 023318).

[0051] As used herein, the term "L19" refers to any antibody that binds to EDB fibronectin or any portion thereof and comprises an amino acid sequence having at least 75% identity to one or more of the following amino acid sequences:

[0052] [Table 1]

[0053] As used herein, the term "VH" refers to the heavy chain variable domain of an antibody.

[0054] As used herein, the term "VL" refers to the light chain variable domain of an antibody.

[0055] As used herein, the terms "CDR1", "CDR2" and "CDR3" refer to the complementarity determining regions within the antibody light or heavy chain variable domain.

[0056] According to some embodiments, there is provided a pharmaceutical composition, dosage form, combination or kit as described above (for the manufacture of a medicament comprising at least one dosage form) for use in treating a human or mammalian patient (i) who has been diagnosed with cancer, (ii) who is suffering from cancer, or (iii) who is at risk of developing cancer.

[0057] This language is to be regarded as embracing both the Swiss-type claim language accepted in some countries (in which case the brackets are to be regarded as not including them) and the EPC 2000 language (in which case the brackets and any content within them are to be regarded as not including them).

[0058] According to one aspect of the present invention there is provided a method for treating a human or mammalian patient, the method comprising administration of an effective amount of one or more of the pharmaceutical compositions, dosage forms, combinations or kits according to the above description.

[0059] According to one embodiment of the method, a human or mammalian patient (i) has been diagnosed with cancer, (ii) is suffering from cancer, or (iii) is at risk of developing cancer.

[0060] According to some embodiments, the cancer is at least one selected from the group consisting of solid or non-solid cancer, malignant lymphoma, liver cancer, lymphoma, leukemia (e.g., acute myeloid leukemia), sarcoma, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colon cancer, cervical cancer, head and neck cancer, esophageal cancer, pancreatic cancer, renal cancer, gastric cancer, and brain cancer.

[0061] According to some embodiments of the pharmaceutical compositions, dosage forms, combinations, kits or methods of treatment described above, the antibody that binds to the fibronectin extra domain B (ED-B) comprises the complementarity determining region (CDR) of the L19 antibody shown in SEQ ID NOs: 10 to 15.

[0062] According to some embodiments of the pharmaceutical compositions, dosage forms, combinations, kits or methods of treatment described above, the antibody that binds to the fibronectin extra domain B (ED-B) comprises the L19 VH shown in SEQ ID NO:7 and / or the L19 VL shown in SEQ ID NO:5.

[0063] According to some embodiments of the pharmaceutical compositions, dosage forms, combinations, kits or methods of treatment described above, the antibody that binds to the fibronectin extra domain B (ED-B) comprises the amino acid sequence of the L19 diabody shown in SEQ ID NO:20.

[0064] According to one embodiment of the pharmaceutical composition, dosage form, combination, kit or method of treatment according to the above description, the kinase inhibitor is a JAK inhibitor.

[0065] Janus kinases (JAKs) are multidomain non-receptor tyrosine kinases that play pivotal roles in intracellular signal transduction. Targeting JAK-related pathways by the use of JAK inhibitors is rapidly entering the clinical arena for a variety of diseases, including myeloproliferative neoplasms, rheumatoid arthritis and other immune-mediated joint diseases, many inflammatory skin diseases, and inflammatory bowel disease.

[0066] In humans, the JAK family includes JAK1 (also known as Janus kinase-1), JAK2 (also known as Janus kinase-2), JAK3 (also known as Janus kinase, leukocyte; JAKL; L-JAK and Janus kinase-3) and TYK2 (also known as protein-tyrosine kinase 2). Regardless of the cytokine receptor, it binds to one or more JAKs to promote signal transduction.

[0067] JAK proteins range in size from 120 to 140 kDa and consist of seven conserved JAK homology (JH) domains; one of which is a functional catalytic kinase domain and one is a pseudokinase domain that may serve regulatory functions and / or serve as a docking site for signal transducers and activators of transcription (STATs).

[0068] STAT recruitment, dimerization, and nuclear translocation result in transcriptional responses. JAK-STAT signaling plays pivotal roles in multiple systems, including the organization and functional capabilities of immune responses, particularly T cell polarization, regulation of hematopoiesis and inflammation, adipogenesis, and proliferation.

[0069] The key role of JAKs in cytokine signaling and therefore in inflammatory diseases such as autoimmune diseases and neoplasms has led to the discovery of therapeutic agents that inhibit the JAK signaling pathway.

[0070] Within the field of hematology, the most rapid clinical expansion of JAK inhibitors has been in the area of ​​myeloproliferative neoplasms.

[0071] The JAK2 Val617Phe mutation, which leads to constitutive activation, is located in the JH2 domain and is found in approximately 60% of myelofibrosis cases, 50-60% of essential thrombocythemia cases, and 97-98% of polycythemia vera cases. Moreover, many myeloproliferative neoplastic diseases, with or without JAK2 mutations, are characterized by increased JAK-STAT signaling via mutations in canonical genes such as CALR.

[0072] In oncology, many solid tumors employ aberrant JAK signaling as part of their pro-survival phenotype and to promote tumor migration.

[0073] Clinical development of JAK inhibitors has progressed rapidly, and in 2011, ruxolitinib (JAK1 and JAK2 inhibitor; Novartis) was approved by the US Food and Drug Administration (FDA) and the European Medicines Agency (EMA) as the first JAK inhibitor for myelofibrosis. Currently approved JAK inhibitors include tofacitinib (Pfizer), fedratinib (Celgene), upadacitinib (AbbVie), peficitinib (Astellas Pharma), and baricitinib (Eli Lilly). Further novel JAK inhibitor compounds are in development, such as ritrecitinib (formerly known as PF-06651600; Pfizer), which acts as a potent inhibitor of JAK3 and tyrosine-protein-kinase TEC (TEC) family kinases (i.e., BTK, BMX, ITK, RLK, and TEC). Clinically relevant agents are listed in the table below.

[0074] Pharmaceutical inhibitors of JAK family members [Table 2]

[0075] According to one embodiment of the pharmaceutical composition, dosage form, combination, kit or method of treatment according to the above description, the JAK inhibitor may be ruxolitinib.

[0076] In particular, ruxolitinib is a small molecule JAK inhibitor used to treat intermediate- or high-risk myelofibrosis, resistant polycythemia vera, and graft-versus-host disease. Ruxolitinib is associated with transient, usually mild, elevations in serum aminotransferases during treatment and can rarely cause self-limited, clinically significant idiopathic acute liver injury, as well as reactivation of hepatitis B in susceptible individuals.

[0077] Ruxolitinib is an orally administered JAK inhibitor with potential antineoplastic and immunomodulatory effects. It is believed to suppress inflammation and cell proliferation by specifically binding to and inhibiting the protein tyrosine kinases JAK 1 and 2.

[0078] Ruxolitinib is a pyrazole substituted at position 1 with a 2-cyano-1-cyclopentylethyl group and at position 3 with a pyrrolo[2,3-d]pyrimidin-4-yl group. It is used as the phosphate salt to treat patients with intermediate- or high-risk myelofibrosis, including primary myelofibrosis, postpolycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis. EXAMPLES

[0079] Example 1 Preparation of Combination Partners 1.1. Ruxolitinib formulations Lyophilized ruxolitinib was first dissolved in 100% DMSO. For in vivo administration, ruxolitinib was prepared at a dose of 75 mg / kg in deionized water containing 5% DMSO + 5% Tween-20.

[0080] 1.2. Protein expression and characterization The L19-IL12 cytokine used in this experiment is a single-chain diabody-type fusion protein consisting of the anti-EDB L19 antibody and a single chain of mouse IL-12, and the βp40 and αp35 subunits of IL-12 are linked by a 15-amino acid linker (GGGGS )3 The cloning of this protein is described in Puca et al. Int J Cancer (2020), 146, 2518-2530.

[0081] 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 protein quality was assessed by SDS-PAGE and size-exclusion chromatography using a Superdex 200 Increase 10 / 300 GL column on an AKTA FPLC.

[0082] 1.3.Results The protein was of high purity and the correct molecular weight.

[0083] Example 2: Therapeutic Experiments 2.1. Preparation of Tumor Cells MC-38 cells derived from C57BL / 6 mouse colon adenocarcinoma cells were cultured in DMEM medium supplemented with fetal bovine serum (10%) and antibiotic-antimycoplasma agent (1%) according to the supplier's protocol, and cultured at 90% confluence for up to 10 passages. Eight-week-old female C57BL / 6 mice were used for the experiment.

[0084] Tumor implantation MC-38 cells were grown to 80% confluence and detached with Trypsin-EDTA 0.05%. Cells were washed, counted, and cultured at a final concentration of 1 × 10 7 cells ml -1 The cells were resuspended in HBSS to a concentration of 1 × 10 6 An aliquot of cells (100 μl of suspension) was injected subcutaneously into the right flank of each animal. Tumor volume was calculated using the following formula: (length × width 2 × 0.5) and measured with a vernier caliper.

[0085] 2.3.Treatment Experiments The average tumor size is about 50-100 mm 3 Once this age had been reached, MC-38 tumor-bearing mice were randomized to start various treatments. Eight different groups (4 / 5 mice per group) received the following treatments: (i) Vehicle was administered intravenously on days 8, 11, and 14 after tumor implantation. (ii) Ruxolitinib (75 mg / kg) was administered subcutaneously on days 8, 11, and 14 after tumor implantation. (iii) L19-IL12 (0.6 mg / kg) was administered intravenously on days 8, 11, and 14 after tumor implantation. (iv) L19-IL12 (0.9 mg / kg) was administered intravenously on days 8, 11, and 14 after tumor implantation. (v) L19-IL12 (1.2 mg / kg) was administered intravenously on days 8, 11, and 14 after tumor implantation. (vi) Ruxolitinib (75 mg / kg) sc and L19-IL12 (0.6 mg / kg) iv on days 8, 11, and 14 after tumor implantation. (vii) Ruxolitinib (75 mg / kg) sc and L19-IL12 (0.9 mg / kg) iv on days 8, 11, and 14 after tumor implantation. (viii) Ruxolitinib (75 mg / kg) sc and L19-IL12 (1.2 mg / kg) iv on days 8, 11, and 14 after tumor implantation Ruxolitinib was administered 10 min before three different doses of L19-IL12, each of which was injected three times into the lateral tail vein 72 h apart.

[0086] The efficacy of the different treatments was monitored by daily measurements of tumor volume using the following formula: Tumor size = (length (mm) x width 2 (mm) / 2.

[0087] The toxicity of the various treatments was monitored by daily body weight measurements and the general appearance of the animals.

[0088] 2.4.Results 2.4.1. Treatment (Figure 2a) L19-IL12 effectively blocked tumor growth both as a monotherapy and in combination with ruxolitinib at each of the three dose levels tested in the study.

[0089] 2.4.2. Toxicity (Figures 2b, 2c, 2d) As expected, administration of L19-IL12 monotherapy caused weight loss at each of the three doses tested in the study, and induced more severe toxicity at the higher doses (1.2 and 0.9 mg / kg) compared to the lowest dose (0.6 mg / kg). Toxic effects were evident on days 14–18.

[0090] However, when L19-IL12 was combined with ruxolitinib, weight loss was minimal at the lower doses (0.6 mg / kg and 0.9 mg / kg) and did not exceed 5% weight loss at the highest dose (1.2 mg / kg).

[0091] Thus, the combination of L19-IL12 and ruxolitinib significantly reduced the toxicity of IL-12.

[0092] Example 3: In vitro bioactivity assays. 3.1. In vitro IFN-γ release Human NK-92 cells were incubated with various inhibitors and subjected to IFN-γ release assay. Baricitinib (HY-15315), tofacitinib (HY-40354), upadacitinib (HY-19569) and fedratinib (HY-10409) were purchased from MedChemExpress LLC. Ruxolitinib (S1378) and dexamethasone (S1322) were purchased from Selleckchem. Prior to the assay, NK-92 cells were starved for 4 hours in plain RPMI medium. Cells were then diluted in complete RPMI at 1x10 6 Cells were resuspended at a density of 100 cells / mL and 100 μL of the cell suspension was incubated with increasing concentrations of inhibitors in the presence of 10 ng / mL L19-hIL12. After 24 h, IFN-γ levels in the culture supernatants were quantified by sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Biolegend).

[0093] 3.2 Results The five JAK inhibitors were benchmarked against dexamethasone, a common anti-inflammatory molecule and commonly used in clinical practice as premedication for cytokine release syndrome (CRS). Surprisingly, all JAK inhibitors, especially ruxolitinib, were superior to dexamethasone in reducing IFN-γ production and release (Figure 5).

[0094] Example 4: High-dose L19-IL2 treatment experiment Tumor cell preparation and tumor implantation were performed as described above in sections 2.1 and 2.2.

[0095] 4.1.Treatment trials The average tumor size is about 50-100 mm 3Once this age had been reached, MC-38 tumor-bearing mice were randomized to start various treatments. Eight different groups (4 / 5 mice per group) received the following treatments: (i) Vehicle was administered intravenously on days 8, 11, and 14 after tumor implantation. (ii) Ruxolitinib (75 mg / kg) sc (subcutaneous injection) on days 8, 11, and 14 after tumor implantation (iii) L19-IL12 (2.4 mg / kg) was administered intravenously on days 8, 11, and 14 after tumor implantation. (iv) L19-IL12 (3 mg / kg) was administered intravenously on days 8, 11, and 14 after tumor implantation. (v) Ruxolitinib (75 mg / kg) sc and L19-IL12 (2.4 mg / kg) iv on days 8, 11, and 14 after tumor implantation (vi) Ruxolitinib (75 mg / kg) sc and L19-IL12 (3 mg / kg) iv on days 8, 11, and 14 after tumor implantation Ruxolitinib was administered 10 minutes before the administration of two different doses of L19-IL12. Each group was injected three times into the lateral tail vein every 72 hours. The efficacy of the different treatments was monitored by measuring the tumor volume daily using the following formula: Tumor size = (length (mm) x width 2 (mm) / 2.

[0096] The toxicity of the various treatments was monitored by daily body weight measurements and the general appearance of the animals.

[0097] 4.2.Results 4.2.1. Treatment (Figure 6) L19-IL12 effectively blocked tumor growth both as a monotherapy and in combination with ruxolitinib at the two highest dose levels tested in the study.

[0098] 2.4.2. Toxicity (continuation of Figure 6) Single-agent administration of L19-IL12 caused severe weight loss (>15%) at the two highest dose levels tested in our experiments, leading to the sacrifice of mice for ethical reasons.

[0099] However, when L19-IL12 was combined with ruxolitinib, weight loss did not exceed 15% and returned to normal by around day 20 after the start of treatment. Thus, even with a high dose of L19-IL12, the combination with ruxolitinib significantly reduced the toxicity of IL-12.

[0100] Summary of results (i) The toxicity of immunocytokines, including IL12, in rodents begins 48 / 72 hours after the second injection (Puca et al. Int J Cancer (2020), 146, 2518-2530). (ii) The half-life of ruxolitinib in rodents is known to be less than 1 hour after injection and in any case is completely eliminated within 24 hours (EMA / 465846 / 2012). (iii) Nevertheless, the combination of L19-IL12 with a kinase inhibitor such as ruxolitinib significantly reduced the toxicity of IL-12. (iv) It is surprising that the kinase inhibitors are no longer present in the blood by the time IL-12 toxicity begins.

[0101] References Puca et al. Int J Cancer (2020), 146, 2518-2530 Neri D, Bicknell R. Tumour vascular targeting.Nat Rev Cancer.2005 Jun;5(6):436-46 Pasche et al. (2011) J Biotechnology, 154, 84-92) Kaspar et al.(2007) Cancer Res, 67, 4940-4948 array The following sequences form part of the disclosure of this application. A WIPO ST25 compatible electronic sequence listing is also attached to this nonprovisional application. For the avoidance of doubt, in the event of any discrepancy between the sequences in the table below and the sequences in the electronic sequence listing, the sequences in the table shall be deemed correct.

[0102]

Table 3

Claims

1. (a) (i) interleukin 12 (IL-12), and (ii) Targeting Entity a targeted immunocytokine comprising (b) kinase inhibitor 23. A pharmaceutical composition comprising:

2. (a) (i) interleukin 12 (IL-12), and (ii) Targeting Entity A recombinant protein comprising (b) kinase inhibitor A dosage form comprising, in a pharma- ceutically acceptable carrier.

3. (a) (i) interleukin 12 (IL-12), and (ii) Targeting Entity A recombinant protein comprising (b) kinase inhibitor A combination including at least

4. (a) (i) interleukin 12 (IL-12), and (ii) Targeting Entity A first dosage form comprising a recombinant protein comprising the compound in a pharma- ceutically acceptable carrier; and (b) a second dosage form comprising a kinase inhibitor in a pharma- ceutically acceptable carrier. A dosage form kit comprising at least:

5. (a) (i) interleukin 12 (IL-12), and (ii) Targeting Entity A recombinant protein comprising (b) kinase inhibitor The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 1 to 4, wherein said

6. (a) (i) interleukin 12 (IL-12), and (ii) Targeting Entity A recombinant protein comprising (b) kinase inhibitor The pharmaceutical composition, dosage form, combination, or kit according to any one of claims 1 to 5, wherein said

7. The pharmaceutical composition, dosage form, combination or kit according to any one of claims 1 to 6, wherein the targeting entity comprises an anti-fibronectin antibody, or a target-binding fragment or derivative thereof.

8. The pharmaceutical composition, dosage form, combination or kit according to any one of claims 1 to 7, wherein the targeting entity comprises an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment or derivative thereof.

9. 9. A pharmaceutical composition, dosage form, combination or kit according to any one of claims 1 to 8 for use in the treatment of a human or mammalian patient (i) diagnosed with cancer, (ii) suffering from cancer, or (iii) at risk of developing cancer (for the manufacture of a medicament comprising at least one dosage form).

10. A method of treating a human or mammalian patient comprising administration of an effective amount of one or more of the pharmaceutical compositions, dosage form combinations or kits according to any one of claims 1 to 8.

11. 11. The method of claim 10, wherein the human or mammalian patient (i) has been diagnosed with cancer, (ii) is suffering from cancer, or (iii) is at risk for developing cancer.

12. The pharmaceutical composition, dosage form, combination, kit or method of treatment according to any one of claims 1 to 11, wherein the cancer is at least one selected from the group consisting of solid or non-solid cancer, malignant lymphoma, liver cancer, lymphoma, leukemia (e.g. acute myeloid leukemia), sarcoma, skin cancer, bladder cancer, breast cancer, uterine cancer, ovarian cancer, prostate cancer, lung cancer, colorectal cancer, cervical cancer, head and neck cancer, esophageal cancer, pancreatic cancer, kidney cancer, gastric cancer and / or brain cancer.

13. An antibody that binds to the extra domain B (ED-B) of fibronectin, a) the complementarity determining regions (CDRs) of the L19 antibody as shown in SEQ ID NOs: 10 to 15, and / or b) L19 VH as shown in SEQ ID NO: 7 and / or L19 VL as shown in SEQ ID NO: 5 The pharmaceutical composition, dosage form, combination, kit or method of treatment according to any one of claims 1 to 12, comprising:

14. The pharmaceutical composition, dosage form, combination, kit or method of treatment according to any one of claims 1 to 13, wherein the antibody that binds to fibronectin extra domain B (ED-B) comprises the amino acid sequence of CDR of L19 shown in SEQ ID NOs: 10 to 15.

15. The pharmaceutical composition, dosage form, combination, kit or method of treatment according to any one of claims 1 to 14, wherein the kinase inhibitor is a JAK inhibitor, preferably ruxolitinib.

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