Compositions Comprising tTF-NGR for Use in Treating Soft Tissue Sarcoma

Abstract

The present invention provides a composition comprising trabectedin or tTF-NGR protein for use in treating cancer in an individual, wherein the treatment comprises the following steps: (a) administering to the individual an effective amount of a composition comprising trabectedin, and (b) subsequently administering to the individual an effective amount of a composition comprising tTF-NGR protein.

Description

[Technical Field]

[0001] The present invention relates to the use of tTF-NGR and trabectedin in the treatment of cancer, particularly soft tissue sarcoma. The inventors surprisingly found that the combination of tTF-NGR and trabectedin results in improved, preferably synergistic, therapeutic activity. Without being bound by theory, the improvement may be due to several effects. Vascular tumor occlusion by tTF-NGR prolongs the intratumoral accumulation of trabectedin, resulting in higher antitumor efficacy. Furthermore, the higher intratumoral procoagulant activity of tTF-NGR may be due to the induction of early apoptosis in tumor cells and tumor endothelial cells by trabectedin, resulting in higher phosphatidylserine (PS) levels in these cells. [Background technology]

[0002] Soft tissue sarcomas (STS) are a rare, heterogeneous group of mesenchymal cancers originating from connective tissue. STS comprise over 100 distinct subtypes, collectively accounting for 1% of all adult cancers. The incidence of STS among sarcomas in the European population ranges between 2 and 5 per 100,000 per year. They arise anywhere in the organism, with common sites being the extremities, trunk, retroperitoneum, and head and neck. Accurate histopathological diagnosis and grading, as well as imaging-based staging, are crucial, as they affect multidisciplinary treatment, which is best performed in experienced reference centers. Due to the heterogeneity and rarity of the disease, diagnosis is often made too late.

[0003] Surgery is the first-line treatment for early-stage and localized STS. However, distant metastases occur in many patients, especially those with high-grade tumors. For patients with unresectable STS, chemotherapy is the standard of care, and this therapeutic approach is multimodal. In cases of metastatic disease, treatment goals are often limited to palliation and / or prolongation of progression-free survival instead of cure. First-line systemic treatment is based on doxorubicin alone or in some combinations, such as doxorubicin and ifosfamide. The combination of gemcitabine and docetaxel appears to be as effective as doxorubicin, but induces more severe hematologic toxicity and is sometimes reserved for second-line treatment. In addition to conventional chemotherapy, trabectedin, pazopanib, and eribulin are alternatives for second-line and further-line treatment (eribulin only in liposarcoma). However, overall survival for patients with advanced STS generally remains poor. Thus, new therapeutic targets and agents in this group of diseases represent an unmet medical need.

[0004] Tumor growth and spread depend on intratumoral angiogenesis to deliver nutrients and oxygen and remove metabolic waste products. Tumor endothelial cells (TECs) are essential for constructing tumor vasculature and reside in the tumor-supportive stroma, surrounded by other cell types (e.g., fibroblasts and some myeloid and immune cells). TECs express a variety of novel targets not present on quiescent endothelial cells in the organism's mature vasculature. Several of these TEC targets are clinically relevant for antiangiogenic therapy in cancer, leading to numerous drugs approved for cancer treatment (e.g., bevacizumab (anti-VEGF moAB), aflibercept (VEGF scavenger), ramucirumab (anti-VEGF-R moAB), sunitinib, sorafenib, and pazopanib (tyrosine kinase inhibitors of TEC tyrosine kinase receptors). The therapeutic activity of these drugs is limited to increasing survival by a few months, and the emergence of resistance limits this approach.

[0005] Conceptually distinct from antiangiogenic treatments is the antivascular approach. Antiangiogenic drugs not only disrupt angiogenesis but also aim to destroy existing neovascularization in tumors. Denekamp et al. proposed existing tumor blood vessels and tumor endothelial cells as target carriers for antivascular therapy (Denekamp et al., Br. J. Cancer, 1982). Tumor vasculature actually provides a therapeutic target for antiangiogenic therapy, vascular destruction, or vasoocclusion and thrombosis followed by tumor infarction. Huang et al. introduced the concept of tumor vasoocclusion by targeted tissue factor (TF) (Huang et al., Science, 1997). Pasqualini et al. characterized a small NGR (asparagine-glycine-arginine)-containing peptide that binds to aminopeptidase N (APN, also known as CD13) as a tumor vascular target (Pasqualini et al., Cancer Res., 2000). CD13 has been shown to promote angiogenesis, tumor growth, and metastasis (Guzman-Rojas et al., Proc. Natl. Acad. Sci., 2012) and has also been shown to be associated with prognosis in patients with some, but not all, cancer histologies examined (Tokuhara et al., Clin. Cancer Res., 2006).

[0006] Trabectedin is a tetrahydroisoquinoline alkaloid available as Yondelis® from Pharma Mar (Madrid, Spain). Trabectedin was originally extracted from the Caribbean sea tunicate Ecteinascidia turbinata but is now synthetically produced and registered in Europe and other countries for the treatment of adult patients with advanced soft tissue sarcoma (Germano et al., Cancer Cell. 2013;23(2):249-262). Trabectedin acts as a DNA binder, binding to the DNA double helix and causing disruption of several transcription factors, DNA-binding proteins, and DNA repair pathways, resulting in G2 / M cell cycle arrest and ultimately apoptosis. Trabectedin has also been found to provide a therapeutic option for metastatic liposarcoma and leiomyosarcoma, as well as a promising candidate for the treatment of synovial sarcoma and high-grade undifferentiated pleomorphic sarcoma (De Santis et al., Drug Design, Develop, and Therapy, 9:5785-5791, 2015).

[0007] However, thrombogenic vascular-targeting agents could theoretically cause serious systemic side effects, such as pulmonary embolism or stroke, etc. Therefore, developing safe and effective dosing regimens for the therapeutic use of vascular-targeting agents is of paramount preclinical and clinical importance. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] Denekamp et al., Br.J.Cancer, 1982 [Non-patent document 2] Huang et al., Science, 1997 [Non-patent document 3] Pasqualini et al., Cancer Res., 2000 [Non-patent document 4] Guzman-Rojas et al., Proc. Natl. Acad. Sci., 2012 [Non-patent document 5] Tokuhara et al., Clin. Cancer Res., 2006 [Non-patent document 6] Germano et al., Cancer Cell.2013;23(2):249-262 [Non-Patent Document 7] De Santis et al., Drug Design, Development. and Therapy, 9:5785-5791, 2015 Summary of the Invention

[0009] It has now surprisingly been found that the combination of tTF-NGR and trabectedin results in improved therapeutic activity, which is preferably synergistic.

[0010] The present invention provides a composition comprising trabectedin for use in the treatment of cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) then administering to said individual an effective amount of a composition comprising tTF-NGR protein. The present invention provides a composition comprising:

[0011] The present invention also provides a composition comprising a tTF-NGR protein for use in treating cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) then administering to the individual an effective amount of a composition comprising the tTF-NGR protein. The present invention provides a composition comprising:

[0012] In one embodiment, the individual's cancer is inoperable, metastatic, or refractory.

[0013] In a further embodiment, the cancer is a soft tissue sarcoma, preferably selected from the group consisting of dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, adult fibrosarcoma, myxofibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma, and undifferentiated sarcoma.

[0014] In another embodiment, the composition comprising trabectedin and the composition comprising tTF-NGR are administered to an individual by intravenous infusion.

[0015] In yet another embodiment, the composition comprising trabectedin is administered to an individual by 24-hour intravenous infusion. Preferably, trabectedin is administered at a dose of 1.5 mg / m 2 is administered at a dose of

[0016] In a preferred embodiment, the tTF-NGR protein comprises or has the sequence of SEQ ID NO: 2. In an alternative embodiment, the tTF-NGR protein has the sequence of SEQ ID NO: 1. SEQ ID NO: 2 comprises SEQ ID NO: 1 and an N-terminal His-tag comprising the N-terminal 46 amino acid residues. While the N-terminal His-tag was retained in the protein used in the examples below, it is understood that it is not necessary for the activity of the protein. SEQ ID NO: 1 and SEQ ID NO: 2 further comprise the sequence of tTF, i.e., amino acids 47 to 264 of SEQ ID NO: 2, and the NGR sequence, i.e., amino acids 265 to 271 of SEQ ID NO: 2.

[0017] In another embodiment, the initial administration of tTF-NGR protein is initiated between one minute and one hour after the end of trabectedin administration. Preferably, the tTF-NGR protein is administered to the individual via a one-hour intravenous infusion. More preferably, the administration of tTF-NGR protein is repeated once daily for four consecutive days, beginning the day after trabectedin administration.

[0018] In a further embodiment, the tTF-NGR protein is at 3 mg / m 2 / day. Preferably, the tTF-NGR protein is administered in a 0.9% NaCl solution with a total infusion volume of 100 ml. More preferably, the tTF-NGR protein is infused via central venous port access. [Brief explanation of the drawings]

[0019] [Figure 1] Illustrative schematic structure of the lead compound tTF-NGR, which contains an N-terminal His tag for detection and purification, full-length tTF, and a targeting peptide with an NGR motif at the C-terminus. [Figure 2] Illustration of targeting and accumulation of TF activity to tumor vasculature and induction of tumor vascular thrombosis and infarction, leading to tumor cell death. A, Components of tumor vascular infarction at the start of treatment and B, Components of tumor vascular infarction during the tumor vascular clotting period. FXa, activated factor X (light blue); FVIIa, activated factor VII (blue). [Figure 3] Demonstration of the therapeutic activity and mode of action of tTF-NGR using different imaging techniques. 1. Bluish coloration of tumors several hours after tTF-NGR injection, visually visualizing blood pooling and vascular destruction (A, left: tTF-NGR; A, right: vehicle control). 2. Intravascular contrast-enhanced magnetic resonance imaging (MRI) of tumors showing tumor anatomy (A, B) and a dramatic decrease in blood flow from high (C) to low (D) blood flow several hours after tTF-NGR application. The vascular volume fraction was quantified as a >1-log decrease. 3. H&E staining of sarcoma xenograft tissue (A, thrombus formation, blood pooling, and vascular destruction after tTF-NGR; B, saline control with normal vasculature). 4. In vivo fluorescence reflectance imaging (B) demonstrating vascular anatomy with intravascular fluorescent fibrin after tTF-NGR. A, before tTF-NGR; B, 1 hour after tTF-NGR; C, vascular destruction 24 hours after tTF-NGR. [Figure 4]Demonstration of the synergistic activity of tTF-NGR and trabectedin in human vascular endothelial cells (HUVECs). A, An example of a flow cytometry experiment showing trabectedin-induced PS upregulation on the HUVEC cell membrane surface. PS is detected by fluorescent PS-binding annexin V-FITC. PS-positive cells are shown in the lower right quadrant (LR) and increase from 7.21% without trabectedin to 29.22% with trabectedin (15 nM, 8-hour incubation). B, Evaluation of seven different experiments with different doses of trabectedin, in which increasing doses of trabectedin induce PS upregulation on the HUVEC surface at 8 and 12 hours (P values ​​are less than 0.0001 for all time points compared to the non-trabectedin control (NTC)). Propidium iodide was used as an internal necrosis control. C, Trabectedin-dependent increase in procoagulant activity of HUVECs upon binding of tTF-NGR (black, without trabectedin; gray, with trabectedin (10 nM, 8 h)), and complete abrogation of this effect by masking PS with different doses of annexin V. Mean + standard error and p-values ​​are from three experiments, each with at least quadruplicate assays. [Figure 5] Illustration of the synergistic activity of tTF-NGR and trabectedin in HT1080 human sarcoma cells. A, Evaluation of six different experiments with 10 nM trabectedin (gray) and an 8-hour incubation time shows significant PS upregulation on the surface of HT1080 sarcoma cells compared to the no-trabectedin control (black). Mean + standard error (p-value = 0.001). Propidium iodide was used as an internal necrosis control. B, Trabectedin-dependent increase in the procoagulant activity of HT1080 sarcoma cells upon binding of tTF-NGR (black, without trabectedin; gray, with trabectedin (10 nM, 8 hours)), and complete abrogation of this effect by masking PS with different doses of Annexin V. Mean + standard error and p-value of four experiments. [Figure 6]In vivo treatment results of trabectedin in combination with tTF-NGR in the HT1080 STS xenograft model. Control, PBS iv; tTF-NGR, 1 mg / kg, day 0; trabectedin, 0.1 mg / kg, day 0; combination, trabectedin + tTF-NGR at the same dose and time schedule (5 hours after trabectedin) (control, n=8; tTF-NGR, n=9; trabectedin, n=9; combination, n=11). DETAILED DESCRIPTION OF THE INVENTION

[0020] Antivascular compounds in the form of bifunctional molecules that target specific structures on the TEC surface with one moiety and carry an antitumor payload as a second moiety are known in the prior art. This class of vascular-targeting compounds aims to target not just one, but two or more tumor types via the common tumor vasculature. Various drugs that target tumor cells containing only one histological entity have been approved for treating specific tumors: for example, moAbs against tumor cell-associated molecules that carry toxins (brentuximab vedotin (CD30+ Hodgkin's disease), trastuzumab emtansine (HER2+ breast cancer), etc.).

[0021] Another class of fusion proteins, designated tTF-NGR, has been developed, in which the nonspecific membrane anchor of tissue factor (TF) is replaced by an NGR motif that binds to CD13. CD13 is an aminopeptidase that is selectively present on the surface of stimulated growing ECs, such as TECs, but is less expressed in other tissues (http: / / www.proteinatlas.org / ENSG00000166825-ANPEP / tissue). Furthermore, CD13 expression is present in some normal tissues (e.g., small bile ducts). However, this expression does not preclude the application of CD13-targeted TF, as this molecule is active only in clotting-competent locations, such as blood vessels, and not elsewhere. This fusion protein (Figure 1) targeted and accumulated TF activity in tumor vasculature, inducing thrombosis and infarction of tumor vessels, leading to tumor cell death (Figure 2).

[0022] These fusion proteins were tested in vitro and in vivo for essential therapeutic properties, such as procoagulant activity, specific binding to their respective target molecules on the surface of stimulated endothelial cells (ECs) or pericytes, in vivo intratumor accumulation, in vivo intratumor activation of coagulation, tumor vasculature occlusion and blood flow inhibition, pharmacodynamic properties including therapeutic antitumor activity in xenografts of human tumors from different histological origins, and finally, safety and toxicology in rodent as well as non-rodent animals. As demonstrated in the experimental results, while retaining full procoagulant activity, tTF-NGR specifically binds to CD13 on the surface of growing ECs, causing tumor vasculature occlusion and infarction, resulting in tumor growth inhibition and regression (Figure 3). The preclinical therapeutic activity of tTF-NGR was independent of tumor histology (e.g., melanoma, lung, breast, sarcoma, glioblastoma). Repeated treatments did not demonstrate any development of resistance.

[0023] Apart from the various specific advantages of tTF-NGR, the present invention has surprisingly found that the combination of tTF-NGR and trabectedin can exhibit synergistic therapeutic activity. Trabectedin is an anti-neoplastic drug originally isolated from the sea squirt Ecteinascidia turbinata. This drug exerts its anti-neoplastic activity by binding to the minor groove of replicating DNA and causing double-strand breaks in the double helix. Furthermore, trabectedin has been found to have a multifaceted mechanism of action in regulating inflammatory mediators in the tumor microenvironment. This effect is likely achieved through selective inhibition of the production of pro-inflammatory cytokines and chemokines (e.g., interleukin-6 (IL-6)), chemokine ligand 2 (CCL2), the matrix binder protein pentraxin 3 (PTX3), and vascular endothelial growth factor (VEGF). In addition, trabectedin depletes macrophages in tumor tissue, and macrophage targeting appears to be an important component of its antineoplastic activity. Trabectedin as a single agent is commonly used as a standard second-line treatment for metastatic or refractory STS. In combination with tTF-NGR, as demonstrated in this invention, both drugs demonstrated enhanced anti-sarcoma activity in xenograft models compared with standard trabectin or tTF-NGR monotherapy. Given the diverse modes of action of trabectedin on tumor cells and different cellular and molecular components of the tumor stroma, the combinatorial therapeutic efficacy of trabectedin in combination with tTF-NGR was unexpected. tTF-NGR has demonstrated preclinical activity in human STS xenografts by occluding and occluding tumor vessels, and the antitumor activity of trabectedin can be improved in these models in vivo by supplementing trabectedin intratumorally, resulting in stronger and longer-lasting antitumor activity. Conversely, trabectedin enhances the procoagulant potency of tTF-NGR in tumor vasculature.The binding site of tTF-NGR, i.e., CD13, is strongly expressed in the vasculature of human STS and / or on the surface of tumor cells in human STS, providing a biomarker for patient selection for this combination that can be further investigated for predictive value of combination treatment activity.

[0024] The present invention further provides clinical dosing regimens for using tTF-NGR and trabectedin in combination therapy to treat cancer patients, particularly patients with soft tissue sarcoma.

[0025] The present invention provides a composition comprising trabectedin for use in the treatment of cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) subsequently administering to said individual an effective amount of a composition comprising a tTF-NGR protein (which preferably comprises or has SEQ ID NO: 2). The present invention provides a composition comprising:

[0026] The present invention also provides a composition comprising a tTF-NGR protein for use in treating cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) subsequently administering to said individual an effective amount of a composition comprising said tTF-NGR protein (which preferably comprises or has SEQ ID NO: 2). The present invention provides a composition comprising:

[0027] In another embodiment, a composition comprising trabectedin and a composition comprising tTF-NGR (which preferably comprises or has SEQ ID NO: 2) are administered to an individual by intravenous infusion.

[0028] In one embodiment, the composition comprising trabectedin is administered to an individual by 24-hour intravenous infusion. Preferably, trabectedin is administered at a dose of 1.5 mg / m 2 is administered at a dose of

[0029] In one embodiment, the tTF-NGR protein has the sequence of SEQ ID NO:2.

[0030] The first dose of tTF-NGR protein can be administered within 1 minute to 1 hour after the end of trabectedin administration. Preferably, tTF-NGR protein is administered to an individual via a 1-hour intravenous infusion. More preferably, tTF-NGR protein administration is repeated once daily for four consecutive days, starting the day after trabectedin administration: for example, trabectedin is administered from 8:00 AM on Monday to 8:00 AM on Tuesday, followed by tTF-NGR by 9:00 AM on Tuesday and the following days (with the final tTF-NGR administered on Friday).

[0031] In a further embodiment, the tTF-NGR protein is at 3 mg / m 2 / day. Preferably, the tTF-NGR protein is administered in a 0.9% NaCl solution with a total infusion volume of 100 ml. More preferably, the tTF-NGR protein is infused via central venous port access.

[0032] Therefore, in a preferred embodiment, the present invention provides a composition comprising trabectedin or tTF-NGR protein for use in the treatment of cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) then administering to said individual an effective amount of a composition comprising said tTF-NGR protein having SEQ ID NO:2. Including, trabectedin is administered to the individual by 24-hour intravenous infusion; and the first administration of the tTF-NGR protein is initiated between 1 minute and 1 hour after the end of the administration of trabectedin; and the tTF-NGR protein is administered to the individual by intravenous infusion for one hour; and the administration of the tTF-NGR protein is repeated once daily for four consecutive days starting the day after the administration of trabectedin; A composition is provided.

[0033] Preferably, the treatment cycle length is 3 weeks. Patients are treated in repeated cycles until there is clear disease progression (iRECIST; Seymour L, Lancet Oncol. 2017), unless other discontinuation criteria exist, and unless either the patient or the investigator requests discontinuation of treatment.

[0034] In one embodiment, the individual's cancer is inoperable, metastatic, or refractory. In a further embodiment, the cancer is a soft tissue sarcoma, preferably selected from the group consisting of dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, adult fibrosarcoma, myxofibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma, and undifferentiated sarcoma.

[0035] Unless otherwise indicated, for purposes of this application, the following terms are intended to have the meanings indicated below as used in the specification and claims.

[0036] "Patient" refers to a mammal, preferably a human. In some embodiments, the patient is one who has failed standard first-line treatment or is unsuitable for standard treatment.

[0037] A "therapeutically effective amount" means the amount of a compound that, when administered to a subject for treating a disease state, is sufficient to effect such improvement in the disease state.

[0038] "Trabectedin" refers to a tetrahydroisoquinoline alkaloid (CAS Registry Number (Chemical Abstracts Service) 0114899-77-3) that is described in the literature (e.g., Germano et al., Cancer Cell. 2013;23(2):249-262) and available as Yondelis® from Pharma Mar (Madrid, Spain).

[0039] "Treatment" means any administration of a therapeutically effective amount of a compound, including: inhibiting a disease in a human experiencing or exhibiting disease pathology or symptomology (i.e., slowing the further development of the pathology and / or symptomology); or • Ameliorating disease in humans experiencing or manifesting disease pathology or symptomology (i.e., reversing the pathology and / or symptomology).

[0040] "iRECIST" refers to a series of modifications to the Solid Tumor Response Evaluation Criteria (RECIST version 1.1) that account for apparent tumor growth that can occur before tumor burden begins to decrease, a phenomenon known as pseudoprogression. These responses occur in a small but significant proportion of patients receiving checkpoint inhibitors and other immunomodulatory agents. The iRECIST guidelines (Seymour L, Lancet Oncol. 2017) are being developed by a team led by Lesley Seymour, MD, PhD, an oncologist at the Canadian Cancer Trials Group based at Queen's University in Kingston, Ontario.

[0041] "PFS" refers to progression-free survival according to iRECIST at 15 weeks (i.e., after 5 cycles), then at 24, 33, 42, and 51 weeks (i.e., every 9 weeks or after 3 cycles adjusted to a 3-week cycle length), and then every 3 months. The reason for using the iRECIST modification of RECIST for evaluation is the observation in preclinical studies and clinical cases that intratumoral swelling due to blood pooling and vascular destruction occurs when tTF-NGR is administered, which can cause pseudoprogressions.

[0042] "OS" indicates overall survival at 12 and 18 months.

[0043] "CR" indicates complete response, which means disappearance of all target lesions. Any pathological lymph nodes (whether target or non-target) must have decreased in the short axis by less than 10 mm.

[0044] "PR" indicates partial response, meaning at least a 30% reduction in the sum of the diameters of the target lesions, using the baseline sum diameter as reference.

[0045] "PD" indicates progressive disease, which means at least a 20% increase in the sum of the diameters of the target lesions, using the smallest sum in the study (which includes the baseline sum if it is the smallest in the study) as the reference. In addition to the 20% relative increase, the sum must also demonstrate an absolute increase of at least 5 mm. (Note: The appearance of one or more new lesions is also considered progression, and rules such as those described in Section 6.1.1 must be observed before progression can be used to terminate treatment in a patient.

[0046] "SD" indicates stable disease, which means that there is not enough shrinkage to qualify for PR or sufficient increase to qualify for PD, using the smallest summated diameter as the reference in the study.

[0047] "TF" denotes tissue factor, ie the known tissue factor protein described in the prior art, for example in Huang et al., Science, 1997.

[0048] "tTF" refers to the known truncated active form of amino acids 33-251 of the tissue factor protein, also described in Huang et al., Science, 1997. The tTF sequence is encompassed by the fusion protein sequences of SEQ ID NO: 1 and SEQ ID NO: 2 (amino acids 47-264 of SEQ ID NO: 2).

[0049] Example 1. Improved therapeutic activity of tTF-NGR and trabectedin The scientific hypothesis behind the combination of tTF-NGR and trabectedin is that the proapoptotic activity of trabectedin on human vascular endothelial cells (HUVECs) and similarly on tumor cells is due to a significant increase in the presence of phosphatidylserine (PS) in the outer leaflet of the phospholipid bilayer that constitutes the cell membrane (see Figures 4A and 4B for HUVECs as an example). This optimized phospholipid environment in the outer cell membrane enhances the procoagulant potency of tTF-NGR within the tTF-NGR:Factor VIIa:Factor X complex on the cell membrane surface (Figure 4C). This effect is specifically dependent on the presence of PS, as it can be completely abolished by masking it with annexin V preincubation.

[0050] Similar experiments were also performed using HT1080 human sarcoma cells instead of HUVECs, since in tumor tissue part of the inner vascular cell layer of the neovasculature is not formed by endothelial cells but is also formed by tumor cells, called "vascular mimics" (see Figure 5).

[0051] To exploit the improved activity in both directions, the combination of trabectedin with tTF-NGR was investigated using a pharmacokinetic approach by applying tTF-NGR approximately 5 hours after trabectedin injection. Results show a significant improvement in therapeutic activity compared to the two individual drugs in the human STS xenograft model HT1080 (Figure 6).

[0052] Example 2. Phase I clinical study of tTF-NGR tTF-NGR was produced in Escherichia coli using a clinical-grade manufacturing process and four-step HPLC purification. The manufacturing process and GMP facilities were approved by the local government (manufacturer's authorization by the Regional Council (Regierungsprasidium)) and the federal Paul Ehrlich Institute (PEI). Toxicity assessments in mice, rats, guinea pigs, and dogs according to EU guidelines S6 and S9 have been completed. In the most sensitive species (mice), the limiting toxicity is pulmonary embolism; in this species, tTF-NGR has a therapeutic safety margin of 1:4 (therapeutic dose:LD10 dose = 1:5).

[0053] An overview of the phase I clinical studies on tTF-NGR is shown in Table 1.

[0054] [Table 1]

[0055] A phase I clinical trial (EudraCT number: 2016-003042-85; NCT02902237) was conducted in patients with late-stage cancer, in which tTF-NGR was administered as a 1-hour infusion in 0.9% NaCl via a central venous line. This study was a first-in-class study guided by repeated contrast-enhanced ultrasound (CEUS) and MRI to assess hypoperfusion in tumor signal lesions. A "5-day q day 22" (5 days daily, 22 days) schedule was used in the study design.

[0056] 4 mg / m 2 At 5 mg / m² / day, dose-limiting toxicity (DLT) was observed due to a transient increase in troponin T without any clinical sequelae. This sensitive laboratory DLT was rapidly reversible, allowing early monitoring for subsequent dose adjustments in future studies. Additionally, one CTCAE grade II deep vein thrombosis of the lower extremities (one patient, completely resolved) also occurred at 5 mg / m². 2 and central venous catheter-related venous thrombosis was observed at 4 mg / m 2 (CTCAE grade II, resolved) and a transient ischemic attack (CTCAE II, resolved) in one patient with angiosarcoma in the left atrium of the heart was observed at 3 mg / m 2 These are events that are reasonably related to tTF-NGR, but are not necessarily caused solely by tTF-NGR. Therefore, the study is based on a recommended dose of 3 mg / m for Phase II (RPIID). 2 The study was completed by administering 5 doses / day (qd 22). The study medication (IMP) was given via PORT central venous access in 100 mL of 0.9% NaCl as a rate-controlled infusion with a duration of 1 hour.

[0057] Pharmacokinetic studies in this study showed that the mean elimination half-life of tTF-NGR was 8.99 hours, and no accumulation was observed with repeated cycles, as pretreatment levels were always reached before the next cycle began.

[0058] Specific inhibition of tumor blood flow (intratumoral blood circulation) was observed in patients measurable by CEUS and MRI without concomitant reduction in blood flow in normal organs. Tumor blood flow inhibition was up to 1-log step. Furthermore, in some metastases, rapid development of areas was observed by MRI, which was interpreted as areas of intratumoral hemorrhage and necrosis, similar to observations in xenograft models. No complete response or partial response (CR) was observed in any of the patients treated to date. However, two patients experienced stable disease (SD) for several months after treatment. Human anti-fusion protein antibodies (HAFA) were detected in a small number of patients when correlated with tumor blood flow inhibition on MRI or CEUS without clinical symptoms (anaphylactic or anaphylactoid reactions) or evidence of neutralizing activity.

[0059] In conclusion, tTF-NGR can be safely applied, and the phase I observations provide proof-of-principle for tumor blood flow inhibition in the clinical setting and a promising therapeutic window. Furthermore, numerous effective strategies exist to theoretically offset the resulting systemic toxicity (heparin, COX inhibitors, dual platelet inhibition with aspirin and P2Y12 inhibitors, fibrinolysis).

[0060] Example 3. Clinical study design to test the anti-sarcoma activity of tTF-NGR in combination with trabectedin.

[0061] 3.1 Research design The study design consisted of a phase II / III, open-label, randomized, controlled study in subjects with or without metastatic or refractory soft tissue sarcoma, with a total recruitment period of 36 months.

[0062] Study participants were patients between 18 and 75 years of age with advanced or metastatic soft tissue sarcoma after unsuccessful first-line anthracycline-containing therapy or with contraindications to these drugs. Patients must have histologic evidence of high-grade, advanced, unresectable, or metastatic soft tissue sarcoma (grade 2–3) according to the FNCLCC grading system, including the following tumor types: dedifferentiated liposarcoma, myxoid liposarcoma (high-grade), pleomorphic liposarcoma, adult fibrosarcoma, myxofibrosarcoma (high-grade), leiomyosarcoma, rhabdomyosarcoma (alveolar, pleomorphic), angiosarcoma, synovial sarcoma, and undifferentiated sarcoma. CD13 positivity on central histology (grade 1+; Kessler T et al., Translational Oncology, 2018) was a prerequisite for study enrollment. Participants must have at least one unidimensionally measurable lesion by computed tomography as defined by iRECIST (Response Evaluation Criteria in Solid Tumors; Seymour L, Lancet Oncol. 2017) criterion 1.1. This lesion must not have been irradiated during previous treatment. Participants are also required to have an ECOG score of ≤2, a life expectancy of at least 3 months, and no adverse reactions to trabectedin. 120 evaluable patients will be enrolled and assigned in parallel in a 1:1 fashion to one of two different arms, as outlined below. Randomization will be stratified by CD13 3+ versus a composite score of CD13 1+ / 2+.

[0063] The study is divided into two parts to be carried out in sequence:

[0064] Phase II part: Prior to the randomized Phase III portion of the study, the combination outlined in Arm 2 (see below, 1.5 mg / m 2 of trabectedin + 3mg / m 2A Phase II safety cohort of six patients receiving at least three cycles of tTF-NGR will be included to confirm the safety of this combination. In the event of dose-limiting toxicity (DLT) in this Phase II cohort, a dose modification protocol to 2 mg / m² for tTF-NGR is planned, and further tapering of tTF-NGR by 0.5 mg / m² is planned in the event of further tolerability issues. Safe doses will then be transferred to the randomized Phase III of the study. The final dose of tTF-NGR proven safe in this combination should be administered to six patients with three cycles each. The randomized (Phase III) portion of the study will begin after safety in the Phase II cohort has been determined by the DSMB.

[0065] Phase III part: In the Phase III portion of this study, 120 patients will be randomized 1:1 into two different arms, with the primary objective to evaluate the efficacy of tTF-NGR in combination with trabectedin, measured as median progression-free survival in the combination treatment arm (arm 2) versus trabectedin monotherapy (arm 1).

[0066] Arm 1: Patients received 1.5 mg / m 2 of trabectedin as a 24-hour central intravenous (iv) infusion on day 1 (qd 22x) until disease progression or further indication (premedication according to institutional guidelines: e.g., 20 mg dexamethasone).

[0067] Arm 2: Patients receive standard trabectedin according to Arm 1, plus 3 mg / m per day for 4 consecutive days after each trabectedin cycle (with an interval of ≤1 hour between the end of trabectedin infusion and tTF-NGR). 2tTF-NGR (1-hour rate-controlled infusion, PORT central venous access, 100 mL NaCl) qd 22x until disease progression or contraindication to further administration: e.g., trabectedin from 8 AM Monday to 8 AM Tuesday, followed by tTF-NGR at 9 AM Tuesday and the following day (final tTF-NGR on Friday).

[0068] Assessment of study outcomes is based on intention-to-treat, so all patients after randomization are part of the efficacy population as assessed by central iRECIST review after study completion.

[0069] Treatment in both arms can be given on an outpatient basis. Some patients may require hospitalization for 24-hour trabectedin infusion. All patients will receive best supportive care (BSC) according to institutional guidelines.

[0070] Anticancer activity is evaluated clinically at 9 weeks, clinically and by imaging at 15 weeks, then every 9 weeks (adjusted to a 3-week cycle length) until 51 weeks, and every 3 months thereafter. The decision regarding the application of the next cycle at 9 weeks is clinical. Imaging and clinical-based decisions follow, starting at 15 weeks. This procedure is described in detail below. Median PFS, PFS rate (iRECIST), and DCR at 15, 24, 33, 42, and 51 (i.e., every 9 weeks, or after 3 cycles adjusted to a 3-week cycle length), and at 3-month intervals thereafter, are calculated; mOS at 12 and 18 months, OS rate, and ORR.

[0071] 3.2 Safety Assessment Safety assessments will be performed continuously during study participation, including standard laboratory evaluations. The incidence of AEs will be summarized by severity in all patients with at least one dose of study drug.

[0072] Patients will be treated in repeated cycles (in the absence of any other withdrawal criteria) until one of the following criteria is met: 1) Unacceptable toxicity precludes further treatment. 2) Disease exacerbation (exacerbation as described below). 3) The patient or investigator requests that the procedure be discontinued.

[0073] Patients who discontinue study treatment in the absence of progressive disease should not receive further cancer therapy of choice until their disease has progressed, unless there is a clear reason for continuing on alternative therapy.

[0074] The administration of the next treatment cycle may be postponed for up to 21 days for the following reasons: 1) because of the patient's clinical condition, or 2) By decision of the investigator after discussion with the principal investigator, Professor Schliemann.

[0075] Detailed follow-up visits after treatment initiation will be performed at weeks 9 and 15, then every 9 weeks (adjusted for a 3-week cycle length) until week 51, and then at 3-monthly intervals thereafter.

[0076] 3.3 Evaluation of the anticancer activity of the combination of tTF-NGR and trabectedin In this study, a modified iRECIST will be used to assess progression-free survival and progression-free survival (PFS), similar to studies using checkpoint inhibitors (Seymour L, Lancet Oncol. 2017). The final iRECIST assessment of PFS and comparison between both arms will be performed in a blinded manner by an independent imaging core laboratory (Prof. M. Wildgruber) at the Department of Radiology (LMU, Munich) after study completion. Ad hoc iRECIST assessments to determine a safe EOT for individual patients will be performed by local investigators at the local study center.

[0077] Anticancer activity (iRECIST modified) will be assessed (clinically and by imaging) at weeks 9 and 15, then every 9 weeks (adjusted for a 3-week cycle length) until week 51, and every 3 months thereafter.

[0078] Overall response will be assessed by analogy with RECIST criteria version 1.1. Complete response (CR): Disappearance of all target lesions. All pathological lymph nodes (whether target or non-target) must have decreased by less than 10 mm in the short axis. • Partial response (PR): At least a 30% reduction in the sum of the diameters of the target lesions, using the baseline sum diameter as reference. Progressive Disease (PD): At least a 20% increase in the sum of the diameters of the target lesions, using the smallest sum in the study (this includes the baseline sum if it is the smallest in the study) as the reference. In addition to the 20% relative increase, the sum must also demonstrate an absolute increase of at least 5 mm. • Stable disease (SD): Neither sufficient shrinkage to qualify for PR nor sufficient increase to qualify for PD, using the smallest summed diameter as the reference in the study.

[0079] Lymph nodes identified as target lesions must always have an actual recorded short-axis measurement (measured in the same anatomical plane as the baseline examination), even if they regress to less than 10 mm in the study. This means that when a lymph node is included as a target lesion, the "sum" of the lesion does not have to be zero, even if the CR criteria are met, because a normal lymph node is defined as having a short axis less than 10 mm. Therefore, the CRF or other data collection method may be designed so that target nodal lesions are recorded in separate sections, where each node must achieve a short axis less than 10 mm to qualify for CR. For PR, SD, and PD, the actual short-axis measurement of the lymph node will be included in the target lesion sum.

[0080] 3.4 Assessment of median progression-free survival (mPFS) and progression-free survival rate PFS duration will be assessed for all randomized subjects. This duration is defined as from randomization until progression or death from any cause. Median PFS (mPFS) will be calculated. PFS rates according to iRECIST will be assessed at 15 weeks (after 5 cycles), then at 24, 33, 42, and 51 weeks (i.e., every 9 weeks, or after 3 cycles adjusted to a 3-week cycle length), and at 3-month intervals thereafter.

[0081] 3.5 Assessment of median overall survival (mOS) and overall survival rate OS will be assessed for all randomized patients. This duration is defined as the time from randomization until death from any cause. Median OS and OS rates at 12 and 18 months will be calculated. Patient survival will then be monitored during regular 3-month follow-up visits.

[0082] References: Denekamp and Hobson, Endothelial-cell proliferation in experimental tumors, Br. J. Cancer (1982) 46, 711 De Santis R, Marrari A, Marchetti S, Mussi C, Balzarini L, Lutman FR, Daolio P, Bastoni S, Bertuzzi AF, Quagliuolo V, Santoro A:Efficacy of trabectedin in advanced soft tissue sarcoma:beyond lipo-and leiomyosarcoma.Drug Design,Development and Therapy 9:5785-5791, 2015 Kessler T、Baumeier A、Brand C、Grau M、Angenendt L、Harrach S、Stalmann U、Schmidt LH、Gosheger G、Hardes J、Andreou D、Dreischaluck J、Lenz G、Wardelmann E、Mesters RM、Schwoppe C、Berdel WE * 、Hartmann W * 、Schliemann C * :Aminopeptidase N(CD13):expression,prognostic impact,and use as therapeutic target for tissue factor induced tumor vascular infarction in soft tissue sarcoma.Translational Oncol.11(6):1271-1282、2018 Guzman-Rojas et al.、Cooperative effects of aminopeptidase N(CD13)expressed by nonmalignant and cancer cells within the tumor microenvironment、PNAS、January 31,2012、109(5)1637-1642 Huang et al.、Tumor Infarction in Mice by Antibody-Directed Targeting of Tissue Factor to Tumor Vasculature、Science、24 Jan 1997:Vol.275、Issue 5299、pp.547-550 Pasqualini et al.、Aminopeptidase N is a Receptor for Tumor-homing Peptides and a Target for Inhibiting Angiogenesis、CANCER RESEARCH 60、722-727、February 1,2000

Claims

1. 1. A composition comprising trabectedin for use in treating cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) subsequently administering to said individual an effective amount of a composition comprising a tTF-NGR protein. A composition comprising:

2. 1. A composition comprising a tTF-NGR protein for use in treating cancer in an individual, said treatment comprising the steps of: (a) administering to said individual an effective amount of a composition comprising trabectedin; and (b) then administering to said individual an effective amount of a composition comprising said tTF-NGR protein. A composition comprising:

3. 3. The composition for use in treating soft tissue sarcoma of claim 1 or claim 2, wherein the cancer in the individual is inoperable, metastatic, or refractory.

4. A composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 2, wherein the cancer is soft tissue sarcoma.

5. The composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 4, wherein the composition comprising trabectedin and the composition comprising tTF-NGR are administered to the individual by intravenous infusion.

6. The composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 5, wherein the composition comprising trabectedin is administered to the individual by intravenous infusion over 24 hours.

7. Trabectedin 1.5 mg / m 2 7. The composition for use in the treatment of soft tissue sarcomas according to claim 1, wherein the composition is administered in a dose of

8. The composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 7, wherein said tTF-NGR protein comprises or has SEQ ID NO:

2.

9. 9. The composition for use in the treatment of soft tissue sarcoma according to claim 1, wherein the first administration of the tTF-NGR protein is initiated between 1 minute and 1 hour after the end of the administration of trabectedin.

10. The composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 9, wherein the tTF-NGR protein is administered to said individual by intravenous infusion for 1 hour.

11. The composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 10, wherein the administration of the tTF-NGR protein is repeated once a day for four consecutive days thereafter.

12. The tTF-NGR protein is 3 mg / m 2 12. The composition for use in the treatment of soft tissue sarcomas according to claim 1, wherein the composition is administered in a dose of 0.1 mg / day.

13. The composition for use in the treatment of soft tissue sarcomas according to one of claims 1 to 12, wherein said tTF-NGR protein is administered in a 0.9% NaCl solution in a total injection volume of 100 ml.

14. The composition for use in the treatment of soft tissue sarcoma according to one of claims 1 to 13, wherein said tTF-NGR protein is administered by central venous port access.

15. The composition for use in the treatment of soft tissue sarcoma described in claim 4, wherein the soft tissue sarcoma is selected from the group consisting of dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, adult fibrosarcoma, myxofibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma and undifferentiated sarcoma.

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