Combination of an immunocytokine containing IL-12 and a kinase inhibitor

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 hereby 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 specifically and individually indicated to be incorporated by reference. In the event of a conflict between the teachings of one or more of the documents incorporated herein by reference and the present disclosure, the teachings of this specification shall govern.

Background Art

[0003] Cytokines are important mediators of innate and adaptive immunity. Although many cytokines are being used for the treatment of advanced cancer patients, their administration is generally associated with severe toxicity, hindering dose escalation to therapeutically active regimens and development as anti-cancer agents. To overcome such problems, the use of "immunocytokines" (i.e., cytokines fused to antibodies or antibody fragments) has been proposed, aiming to concentrate the immune system-stimulating activity at the disease site while sparing normal tissues (Neri & Bicknell, 2005). However, even when cytokines are genetically fused to antibodies or antibody fragments to create "immunocytokines," it is not always possible to obtain immunocytokines that retain the ability of the antibody to target tumors. For example, in certain interleukin-7 fusions (Pasche et al. (2011) J Biotechnology, 154, 84-92), tumor targeting was completely abolished, while the tumor targeting ability of certain GM-CSF fusions (Kaspar et al. (2007) Cancer Res, 67,4940-4948) was found to be dose-dependent.

[0004] IL-12 is produced by antigen-presenting cells such as macrophages and CDlc+ dendritic cells, and natural killer (NK) cells, CD8 +Cytotoxic T cells, CD4 + Acts on T helper cells. IL12, originally called natural killer cell stimulatory factor, acts on NK cells and CD8 + T cells to promote cytotoxic activity and promotes polarization of CD4 + T cells to the type 1 phenotype.

[0005] Interestingly, human CD4 + and CD8 + T cells introduced into the heterologous gene environment of IL-12-deficient humanized mice preferentially differentiate into type 2 (IL4+ GATA3+) or mixed type 1 and 2 (IFNG+ TBET+ IL4+ GATA3+) subsets. Injection of recombinant human IL-12 into mice restored differentiation to type 1 and improved cytotoxic immunity against 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 mycobacterial disease, suggesting insufficient type 1 cell-mediated immunity. In mice, genetic deletion of IL-12RB2, another component of the IL-12 receptor, enhances susceptibility to spontaneous autoimmunity, B cell malignancies, and lung cancer.

[0006] As a single agent, intravenous injection of recombinant IL-12 has shown moderate clinical efficacy in a subset of patients with advanced melanoma and renal cell carcinoma. However, due to a death in the first phase I trial from Clostridia perfringens sepsis, interest in systemic administration of IL-12 is limited.

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

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

[0009] In clinical trials involving cancer patients, promising therapeutic activity was revealed, but at the same time, it was also shown that recombinant human IL-12 is extremely toxic to humans, with a maximum tolerated dose of 0.5 μg / kg of body weight.

[0010] Due to the toxic side effects of toxins, especially cytokines such as IL-12, it was difficult to administer an effective amount and reach a high concentration at the tumor site.

[0011] Previously, researchers have tried to overcome these drawbacks, for example, by targeting the delivery of IL-12 cytokine to the tumor environment through binding to an antibody specific to an antigen associated with cancer growth. These cytokine-antibody conjugates are often referred to as "immunocytokines".

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

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

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

[0015] To overcome the drawbacks associated with IL-12 therapy, it has been proposed to deliver IL-12 to the tumor site by an antibody against a tumor-associated marker, increase the local concentration of IL-12 at the tumor site, and reduce 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 neovessels are more accessible to therapeutics administered intravenously than tumor cells, which helps avoid 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 existing blood vessels. Tumors induce angiogenesis by secreting various growth factors (such as vascular endothelial growth factor). Tumor angiogenesis enables tumors to grow to a diameter of several millimeters or more and is also a prerequisite for tumor metastasis. The newly formed blood vessels resulting from angiogenesis form the tumor neovasculature or tumor metastases. Targeting IL-12 to the neovasculature should enable immunotherapy for various different types of tumors.

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

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

[0018] Furthermore, immunocytokines based on L19 are currently being studied in phase I, phase II, and phase III clinical trials for cancer patients. These immunocytokines include several cytokines including IL-12.

Summary of the Invention

Problems to be Solved by the Invention

[0019] One object of the present invention is to expand the therapeutic application range of the identified immunocytokines described above.

[0020] Another object of the present invention is to provide a new treatment method for pathological conditions for which no appropriate treatment method currently exists.

[0021] Another object of the present invention is to improve the effectiveness of treatment using the above immunocytokines.

Brief Description of the Drawings

[0022]

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Mode for Carrying Out the Invention

[0023] The present invention particularly provides a combination of an immunocytokine containing IL-12 and a kinase inhibitor. According to a first aspect of the present invention, (a) (i) Interleukin 12 (IL-12) and (ii) a targeting entity to form a targeted immunocytokine, and (b) a kinase inhibitor are provided in a pharmaceutical composition: The inventors unexpectedly recognized that while the kinase inhibitor reduces the toxicity of the targeted IL-12, its anti-cancer activity remains unchanged.

[0024] In particular, the inventors found that when a targeted immunocytokine containing interleukin-12 (IL-12) is administered to tumor-bearing mice in combination with such a kinase inhibitor, the targeted IL-12 maintains its anti-cancer activity while its toxicity is significantly reduced.

[0025] There was no a priori expectation of reducing the toxicity of IL-12 by combining it with a kinase inhibitor, but as the experimental results showed, the results were even more surprising.

[0026] As used herein, the term "kinase inhibitor" (TKI) refers to compounds that inhibit tyrosine kinases, mainly those of low molecular weight. Tyrosine kinases are enzymes responsible for activating many proteins through signal transduction cascades. Proteins are activated by adding a phosphate group (phosphorylation) to the protein in the step that TKI inhibits. TKI is generally used as an anticancer agent. TKI acts through four different mechanisms: competing with the phosphorylated form of adenosine triphosphate (ATP), the substrate, or both, and acting allosterically, that is, binding to a site other than the active site and being able to affect activity through a conformational change. Recently, it has been shown that TKI deprives the Cdc37-Hsp90 molecular chaperone system, on which tyrosine kinases depend for cell stability, and causes ubiquitination and degradation. Signal transduction therapy can also be used for 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 the cell membrane have the potential to bridge tumor cells and specific leukocytes (e.g., T cells and NK cells), similar to what is achieved using bispecific antibodies. In contrast, immunocytokines targeting extracellular matrix components associated with tumors (e.g., splice isoforms of fibronectin and tenascin C) are thought to exhibit biological activity mainly by the high-density immobilization of the cytokine moiety at the disease site.

[0028] As used herein, the term "targeting entity" refers to a molecule that can bind to a predetermined target with high specificity and affinity. Such targeting entities are, for example, antibodies, or fragments or derivatives thereof.

[0029] Interleukin 12 (IL-12) is an interleukin that is naturally produced from dendritic cells, macrophages, neutrophils, and human B lymphoblastoid cells (NC-37) in response to antigen 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 (referred to as "p70") and the p40 homodimer 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 γ (IFN-γ) and tumor necrosis factor α (TNF-α) from T cells and natural killer (NK) cells, and suppresses the suppression of IFN-γ by IL-4. T cells that produce IL-12 possess a coreceptor called CD30 that is associated with the activity of IL-12.

[0031] IL-12 plays an important role in the activation of natural killer cells and T lymphocytes. IL-12 enhances the cytotoxic activities of NK cells and CD8+ cytotoxic T lymphocytes. There also seems to be a relationship 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 important proteins involved in IL-12 signaling in NK cells. The enhancement of functional responses is shown by IFN-γ production and target cell killing.

[0032] IL-12 also has an anti-angiogenic effect and can inhibit the formation of new blood vessels. This is done by increasing the production of interferon gamma, which in turn increases the production of a chemokine called induced protein-10 (IP-10 or CXCL10). IP-10 mediates this anti-angiogenic effect. Because IL-12 has the ability to induce an immune response and an anti-angiogenic effect, there is interest in testing IL-12 as an anti-cancer agent. However, in the tumors tested to date, no substantial activity has been observed. There may be a therapeutic relevance between IL-12 and psoriasis and inflammatory bowel disease.

[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) a targeting entity comprising a recombinant protein, and (b) a kinase inhibitor There is provided a dosage form comprising the same in a pharmaceutically acceptable carrier.

[0037] According to another aspect of the present invention, at least (a)(i) interleukin 12 (IL-12) and (ii) a targeting entity A recombinant protein comprising, and (b) a kinase inhibitor There is provided a combination comprising.

[0038] According to another aspect of the present invention, at least (a)(i) interleukin 12 (IL-12) and (ii) a targeting entity A first dosage form comprising the recombinant protein comprising in a pharmaceutically acceptable carrier, and (b) a second dosage form comprising a kinase inhibitor in a pharmaceutically acceptable carrier There is provided a kit of dosage forms 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) a targeting entity A recombinant protein comprising: and (b) a 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) a targeting entity A recombinant protein comprising: and (b) a 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 before a 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. Fibronectin also binds to extracellular matrix proteins such as collagen, fibrin, and heparan sulfate proteoglycans (such as syndecan).

[0045] Fibronectin exists as a protein dimer, with two nearly identical monomers linked by a pair of disulfide bonds. The fibronectin protein is produced from a single gene, but several isoforms are created by alternative splicing of its pre-mRNA.

[0046] There are two types of fibronectin in vertebrates: · Soluble plasma fibronectin (formerly called "cold-insoluble globulin", or CIg) is a major protein component in 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 as a dimer of soluble protein from various cells, mainly fibroblasts, and is then assembled into an insoluble matrix through a complex cell-mediated process.

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

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

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

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

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

[0052] [Table 1]

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

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

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

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

[0057] This language is considered to encompass both the Swiss-type claim language (wherein the brackets are considered absent) as accepted in some countries and the language of the EPC2000 (wherein the brackets and the content therein are considered absent).

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

[0059] According to one embodiment of the method, the human or mammalian patient (i) has been diagnosed with cancer, (ii) has 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 cancer 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 brain tumors.

[0061] According to some embodiments of the pharmaceutical composition, dosage form, combination, kit or treatment method according to the above description, an antibody that binds to the extra domain B (ED-B) of fibronectin comprises the complementarity determining regions (CDRs) of the L19 antibody shown in SEQ ID NOs: 10 to 15.

[0062] According to some embodiments of the pharmaceutical composition, dosage form, combination, kit or treatment method according to the above description, an antibody that binds to the extra domain B (ED-B) of fibronectin 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 composition, dosage form, combination, kit or treatment method according to the above description, an antibody that binds to the extra domain B (ED-B) of fibronectin 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 treatment method according to the above description, the kinase inhibitor is a JAK inhibitor.

[0065] Janus kinase (JAK) is a multi-domain non-receptor tyrosine kinase and plays a very important role in intracellular signal transduction. Targeting of JAK-related pathways by the use of JAK inhibitors has rapidly entered the clinical arena for various diseases such as myeloproliferative neoplasms, rheumatoid arthritis and other immune-mediated arthropathies, many inflammatory skin diseases, and inflammatory bowel diseases.

[0066] In humans, the JAK family includes JAK1 (also known as Janus kinase-1), JAK2 (also known as Janus kinase-2), JAK3 (Janus kinase, leukocyte; JAKL; L-JAK and also known as Janus kinase-3) and TYK2 (also known as protein-tyrosine kinase 2). Cytokine receptors, regardless of their type, bind to one or more JAKs to promote signal transduction.

[0067] The JAK proteins are 120 - 140 kDa in size and consist of seven conserved JAK homology (JH) domains; one of these is a functional catalytic kinase domain, another is a pseudokinase domain that may play a regulatory function, and / or a domain that serves as a docking site for signal transducer and activator of transcription (STAT).

[0068] Recruitment, dimerization, and nuclear translocation of STAT trigger transcriptional responses. JAK - STAT signaling plays a critically important role in a wide range of systems, including immune responses, particularly T - cell polarization, regulation of hematopoiesis and inflammation, adipogenesis, and tissue organization and functional capacity such as proliferation.

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

[0070] In the field of hematology, the most significant 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, 50 - 60% of essential thrombocythemia, and 97 - 98% of polycythemia vera. Furthermore, regardless of the presence or absence of JAK2 mutations, many myeloproliferative neoplasm diseases are characterized by enhanced JAK - STAT signaling through mutations in canonical genes such as CALR.

[0072] In the field of oncology, many solid tumors engage in abnormal JAK signaling as part of a phenotype that promotes survival and also to promote tumor migration.

[0073] The clinical development of JAK inhibitors has been progressing rapidly. In 2011, ruxolitinib (a 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). The development of new JAK inhibitor compounds, such as ritlecitinib (previously 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), is also ongoing. Clinically relevant agents are shown 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 treatment method according to the above description, the JAK inhibitor can be ruxolitinib.

[0076] In particular, ruxolitinib is a small molecule JAK inhibitor and is used for the treatment of intermediate-risk or high-risk myelofibrosis, refractory polycythemia vera, and graft-versus-host disease. Ruxolitinib may be accompanied by a transient and usually mild increase in serum aminotransferase during treatment, and rarely cause self-limiting and clinically obvious idiopathic acute liver injury. In addition, it may cause reactivation of hepatitis B in highly sensitive individuals.

[0077] Ruxolitinib is an orally administrable JAK inhibitor, and anti-tumor and immunomodulatory effects are expected. Ruxolitinib is thought to suppress inflammation and cell proliferation by specifically binding to and inhibiting protein tyrosine kinases JAK 1 and 2.

[0078] Ruxolitinib is a pyrazole substituted at the 1-position with a 2-cyano-1-cyclopentylethyl group and at the 3-position with a pyrrolo[2,3-d]pyrimidin-4-yl group. It is used as a phosphate in the treatment of patients with intermediate or high-risk myelofibrosis, such as primary myelofibrosis, post-polycythemia vera myelofibrosis, and post-essential thrombocythemia myelofibrosis.

Example

[0079] Example 1: Preparation of combination partners 1.1. Ruxolitinib formulation 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-form fusion protein in which an anti-EDB L19 antibody is fused with a single chain of mouse IL-12, and the βp40 subunit and αp35 subunit 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 cell culture supernatants by affinity chromatography using a protein A affinity column. After dialysis against PBS pH 7.4, the protein quality was evaluated by size exclusion chromatography using SDS-PAGE and a Superdex 200 Increase 10 / 300 GL column mounted on an AKTA FPLC.

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

[0083] Example 2: Treatment experiment 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 antibiotics-antimycotic agent (1%) according to the protocol of the supplier, and cultured until the confluence was below 90% and up to 10 passages. Female C57BL / 6 mice at 8 weeks of age were used in the experiment.

[0084] 2.2. Tumor transplantation MC-38 cells were grown to 80% confluence and detached with 0.05% Trypsin-EDTA. The cells were washed, counted, and resuspended in HBSS to a final concentration of 1×10 7 cells / ml -1 . An aliquot of 1×10 6 cells (100 μl of the suspension) was subcutaneously injected into the right flank of each animal. The tumor volume was determined by the following formula: (length × width 2 × 0.5) and measured with calipers.

[0085] 2.3. Treatment experiment When the tumors reached an average of approximately 50 - 100 mm 3 , MC-38 tumor-bearing mice were randomly assigned and various treatments were initiated. The following treatments were performed on 8 different groups (4 / 5 mice per group): (i) Vehicle was administered i.v. (intravenous injection) on days 8, 11, and 14 after tumor transplantation (ii) Ruxolitinib (75 mg / kg) was administered s.c. (subcutaneous injection) on days 8, 11, and 14 after tumor transplantation (iii) L19-IL12 (0.6 mg / kg) was administered i.v. on days 8, 11, and 14 after tumor transplantation (iv) L19-IL12 (0.9 mg / kg) was administered i.v. on days 8, 11, and 14 after tumor transplantation (v) L19-IL12 (1.2 mg / kg) was administered i.v. on days 8, 11, and 14 after tumor transplantation (vi) Ruxolitinib (75 mg / kg) was administered s.c. and L19-IL12 (0.6 mg / kg) was administered i.v. on days 8, 11, and 14 after tumor transplantation (vii) On days 8, 11, and 14 after tumor transplantation, loxoribine (75 mg / kg) was administered subcutaneously and L19-IL12 (0.9 mg / kg) was administered intravenously. (viii) On days 8, 11, and 14 after tumor transplantation, loxoribine (75 mg / kg) was administered subcutaneously and L19-IL12 (1.2 mg / kg) was administered intravenously. Loxoribine was administered 10 minutes before administering three different doses of L19-IL12. Each group was injected three times into the lateral tail vein every 72 hours.

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

[0087] The toxicity of 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 at each of the three dose levels tested in the experiment, whether as monotherapy or in combination with loxoribine.

[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 experiment, with higher doses (1.2 and 0.9 mg / kg) causing more severe toxicity compared to the lowest dose (0.6 mg / kg). The toxic effects were prominent on days 14 - 18.

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

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

[0092] Example 3: In vitro bioactivity assay. 3.1. IFN-γ release in vitro Human NK-92 cells were incubated with various inhibitors and an IFN-γ release assay was performed. 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 in plain RPMI medium for 4 hours. Thereafter, the cells were resuspended in complete RPMI at a density of 1x10 6 cells / mL and 100 μL of the cell suspension was incubated with increasing concentrations of the inhibitors in the presence of 10 ng / mL of L19-hIL12. After 24 hours, the IFN-γ levels in the culture supernatant were quantified by sandwich enzyme-linked immunosorbent assay (ELISA) using a commercially available kit (Biolegend).

[0093] 3.2 Results Five JAK inhibitors, which are common anti-inflammatory molecules and are generally benchmarked against dexamethasone, which is commonly used as a pretreatment for cytokine release syndrome (CRS) in clinical practice. Surprisingly, all JAK inhibitors, especially ruxolitinib, were superior to dexamethasone in reducing the production and release of IFN-γ (Figure 5).

[0094] Example 4: Treatment experiment with high-dose L19-IL2 The preparation of tumor cells and the transplantation of tumors were performed as described in Sections 2.1 and 2.2 above.

[0095] 4.1. Treatment experiment Tumors were approximately 50 - 100 mm in mean 3When this was reached, MC-38 tumor-bearing mice were randomly assigned and various treatments were initiated. The following treatments were performed on 8 different groups (4 / 5 mice per group): (i) Vehicle i.v. (intravenous injection) on days 8, 11, and 14 after tumor transplantation (ii) Ruxolitinib (75 mg / kg) s.c. (subcutaneous injection) on days 8, 11, and 14 after tumor transplantation (iii) L19-IL12 (2.4 mg / kg) i.v. on days 8, 11, and 14 after tumor transplantation (iv) L19-IL12 (3 mg / kg) i.v. on days 8, 11, and 14 after tumor transplantation (v) Ruxolitinib (75 mg / kg) s.c. and L19-IL12 (2.4 mg / kg) i.v. on days 8, 11, and 14 after tumor transplantation (vi) Ruxolitinib (75 mg / kg) s.c. and L19-IL12 (3 mg / kg) i.v. on days 8, 11, and 14 after tumor transplantation Ruxolitinib was administered 10 minutes before the administration of the two different doses of L19-IL12. Each group was injected 3 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) × 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 at the two high-dose levels tested in the experiment, either as monotherapy or in combination with ruxolitinib.

[0098] 2.4.2. Toxicity (Continued from Figure 6) Administration of L19-IL12 as a single agent caused severe weight loss (more than 15%) at the two high-dose levels tested in the experiment, resulting in sacrificing the mice for ethical reasons.

[0099] However, when L19-IL12 was combined with ruxolitinib, the weight loss did not exceed 15%, and it returned to normal around the 20th day after the start of treatment. Therefore, 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 containing IL12 in rodents starts 48 / 72 hours after the second injection (Puca et al. Int J Cancer (2020), 146, 2518-2530). (ii) The blood half-life of ruxolitinib in rodents is known to be less than 1 hour after injection and is completely eliminated within 24 hours in any case (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) The fact that the kinase inhibitor is no longer present in the blood when the toxicity of IL-12 begins is surprising.

[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 Sequences The following sequences form part of the disclosure of this application. An electronic sequence listing compatible with WIPO ST25 is also attached to the non-provisional application. To avoid doubt, if there is a discrepancy between the sequences in the following table and those in the electronic sequence listing, the sequences in this table shall be considered correct.

[0102]

Table 3

Claims

1. (a) (i) Interleukin 12 (IL-12) and (ii) a targeting entity comprising an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment thereof a recombinant protein comprising, and (b) a kinase inhibitor at least comprising, a combination for cancer treatment, wherein the kinase inhibitor is the JAK inhibitor ruxolitinib, and the targeting entity is an antibody that can bind to a predetermined target with high specificity and affinity, or a target-binding fragment thereof, a combination for cancer treatment.

2. (a) In a pharmaceutically acceptable carrier (i) Interleukin 12 (IL-12) and (ii) an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment thereof a recombinant protein comprising, and (b) a kinase inhibitor in a pharmaceutically acceptable carrier at least comprising, a kit for cancer treatment, wherein the kinase inhibitor is the JAK inhibitor ruxolitinib, and the targeting entity is an antibody that can bind to a predetermined target with high specificity and affinity, or a target-binding fragment thereof, a kit for cancer treatment.

3. (a) (i) Interleukin 12 (IL-12) and (ii) a targeting entity comprising an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment thereof a targeted immunocytokine comprising, and (b) a kinase inhibitor comprising, a pharmaceutical composition for cancer treatment, wherein the kinase inhibitor is the JAK inhibitor ruxolitinib, and the targeting entity is an antibody that can bind to a predetermined target with high specificity and affinity, or a target-binding fragment thereof, a pharmaceutical composition for cancer treatment.

4. (a) (i) Interleukin 12 (IL-12) and (ii) a targeting entity comprising an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment thereof a recombinant protein comprising, and (b) a kinase inhibitor in a pharmaceutically acceptable carrier, a pharmaceutical for cancer treatment. The kinase inhibitor is the JAK inhibitor ruxolitinib, and the targeting entity is an antibody that can bind to a predetermined target with high specificity and affinity, or a target-binding fragment thereof, a pharmaceutical for cancer treatment.

5. (a) (i) Interleukin 12 (IL-12) and (ii) a targeting entity comprising an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment thereof A recombinant protein comprising, and (b) a kinase inhibitor are simultaneously administered or taken, the pharmaceutical composition, medicine, combination, or kit for cancer treatment according to any one of claims 1 to 4.

6. (a) (i) Interleukin 12 (IL-12) and (ii) a targeting entity comprising an antibody that binds to the extra domain B (ED-B) of fibronectin, or a target-binding fragment thereof A recombinant protein comprising, and (b) a kinase inhibitor are sequentially administered or taken, the pharmaceutical composition, medicine, combination, or kit for cancer treatment according to any one of claims 1 to 4.

7. The antibody that binds to the extra domain B (ED-B) of fibronectin is (a) the complementarity-determining regions (CDRs) of the L19 antibody shown in SEQ ID NOs: 10 to 15, and / or (b) L19 VH shown in SEQ ID NO: 7 and / or L19 VL shown in SEQ ID NO: 5 comprises, the pharmaceutical composition, medicine, combination, or kit for cancer treatment according to any one of claims 1 to 4.

8. The antibody that binds to the extra domain B (ED-B) of fibronectin comprises the amino acid sequences of the CDRs of L19 shown in SEQ ID NOs: 10 to 15, the pharmaceutical composition, medicine, combination, or kit for cancer treatment according to any one of claims 1 to 4.

9. The cancer is at least one selected from the group consisting of solid cancer 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 tumor, the pharmaceutical composition, medicine, combination, or kit for cancer treatment according to any one of claims 1 to 4.

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