Compositions comprising tTF-NGR for 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 steps of: (a) administering an effective amount of a composition comprising trabectedin to the individual, and (b) subsequently administering an effective amount of a composition comprising tTF-NGR protein to the individual.

Description

Technical Field

[0001] The present invention relates to the use of tTF-NGR and Trabectedin in the treatment of cancer, especially soft tissue sarcoma. The inventors were surprised to find that the combination of tTF-NGR and Trabectedin provides an improved, more synergistic therapeutic activity. Without being bound by theory, this improvement can be caused by several effects. TTF-NGR blocking vascular tumors can prolong the accumulation of Trabectedin in the tumor, thereby producing a higher anti-tumor effect. In addition, the higher intratumoral procoagulant activity of tTF-NGR may be caused by Trabectedin inducing early apoptosis of tumor cells and tumor endothelial cells, resulting in higher phosphatidylserine (PS) levels on these cells. Background Art

[0002] Soft tissue sarcomas (STS) are rare, heterogeneous mesenchymal cancers that arise from connective tissue. STS consist of more than 100 different subtypes that collectively account for 1% of all adult cancers, with an incidence of STS in Europa sarcomas ranging from 2-5 / 100,000 / year. They arise anywhere in the organism, with common sites being the extremities, trunk, retroperitoneum, and head / neck. Accurate histopathological diagnosis and grading as well as radiographic tumor staging are important as it influences multidisciplinary treatment and are best performed in experienced reference centers. Due to the heterogeneity and rarity of the disease, diagnosis is often late.

[0003] Surgery is the first line of treatment for early-stage and localized STS. However, many patients develop distant metastases, especially in those with malignant tumors. For patients with unresectable STS, chemotherapy is the standard of care and therapy is multidisciplinary. In cases of metastatic disease, the goal of treatment is usually limited to remission and / or prolonged progression-free survival, rather than cure. First-line systemic therapy is based on doxorubicin alone or in several combinations, such as doxorubicin and ifosfamide. The gemcitabine and docetaxel combination 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 treatment, with eribulin being used only for liposarcoma. However, overall survival of patients with advanced STS remains poor overall. Therefore, there is an unmet need for new therapeutic targets and drugs in this disease category.

[0004] Tumor growth and spread depend on newly formed blood vessels within the tumor to deliver nutrients and oxygen and remove metabolic waste products. Tumor endothelial cells (TECs) are essential for building tumor blood vessels, and other cell types such as fibroblasts and some myeloid and immune cells belong to the tumor-supporting matrix. New targets expressed by TECs are not found in the quiescent endothelial cells of the body's mature vasculature. Some of these TEC targets are clinically relevant for anti-angiogenic therapy of cancer, which has led to the approval of many drugs for cancer treatment (e.g., bevacizumab (anti-VEGF moAB), aflibercept (VEGF trap), ramucirumab (anti-VEGF-RmoAB), sunitinib, sorafenib, pazopanib (examples of tyrosine kinase inhibitors for TEC tyrosine kinase receptors)). The therapeutic activity of these drugs is limited to a few months, and the increase in survival and the development of drug resistance limit this approach.

[0005] Conceptually different from anti-angiogenic therapy is the anti-vascular approach. Anti-vascular drugs not only interfere with angiogenesis, but also aim to destroy new blood vessels that already exist in the tumor. Denekamp et al. proposed existing tumor blood vessels and tumor endothelial cells as target carriers for anti-angiogenic therapy (Denekamp et al., Br. J. Cancer, 1982). The vascular system of the tumor does provide a therapeutic target for anti-angiogenic therapy, vascular destruction or vascular occlusion and subsequent tumor infarction thrombosis. Huang et al. introduced the concept of tumor vascular occlusion by targeting tissue factor (TF) (Huang et al., Science, 1997). Pasqualini et al. combined a peptide containing a small NGR (asparagine-glycine-arginine) with 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 certain but not all histologically examined cancers (Tokuhara et al., Clin. Cancer Res., 2006).

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

[0007] However, thrombotic vascular-targeted drugs may theoretically cause serious systemic side effects, such as pulmonary embolism or stroke. Therefore, developing safe and effective dosing regimens for the therapeutic use of vascular-targeted drugs is of great preclinical and clinical significance. Summary of the invention

[0008] The present invention surprisingly found that the combination of tTF-NGR and Trabectedin provides an improved, more synergistic therapeutic activity.

[0009] The present invention provides a composition containing trabectedin for use in treating cancer in an individual, wherein the treatment comprises the following steps:

[0010] (a) administering to the individual an effective amount of a composition comprising trabectedin, and

[0011] (b) subsequently administering to the individual an effective amount of a composition comprising a tTF-NGR protein.

[0012] The present invention also provides a composition comprising a tTF-NGR protein for use in treating cancer in an individual, wherein the treatment comprises the following steps:

[0013] (a) administering to the individual an effective amount of a composition comprising trabectedin, and

[0014] (b) subsequently administering to the individual an effective amount of a composition comprising a tTF-NGR protein.

[0015] In one embodiment, the cancer in the individual is inoperable, metastatic, or refractory.

[0016] In a further embodiment, the cancer is a soft tissue sarcoma, and is preferably selected from the group consisting of dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, adult fibrosarcoma, myxofibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma, and undifferentiated sarcoma.

[0017] In another embodiment, said composition comprising trabectedin and said composition comprising tTF-NGR are administered to said individual by intravenous infusion.

[0018] In another embodiment, the composition comprising trabectedin is administered to the individual by 24-hour intravenous infusion. The administration dose of trabectedin is preferably 1.5 mg / m 2 .

[0019] In a preferred embodiment, the tTF-NGR protein includes or has the sequence of SEQ ID NO:2. In another embodiment, the tTF-NGR protein has the sequence of SEQ ID NO:1. SEQ ID NO:2 includes SEQ ID NO:1 and an N-terminal His-tag, which includes 46 amino acid residues at the N-terminus. Although the N-terminal His-tag is retained in the proteins used in the following examples, it is understood that this is not necessary for the activity of the protein. SEQ ID NOs:1 and 2 also include the tTF sequence, amino acids 47 to 264 of SEQ ID NO:2 and the NGR sequence, amino acids 265 to 271 of SEQ ID NO:2.

[0020] In another embodiment, the first administration of the tTF-NGR protein is initiated between 1 minute and 1 hour after the end of the administration of trabectedin. Preferably, the tTF-NGR protein is administered to the individual by 1 hour intravenous infusion. More ideally, the administration of the tTF-NGR protein is repeated once a day for 4 consecutive days after the administration of trabectedin.

[0021] In another embodiment, the tTF-NGR protein is administered at 3 mg / m 2 Preferably, the tTF-NGR protein is infused in a 0.9% NaCl solution, with a total infusion volume of 100 ml. More preferably, the tTF-NGR protein is infused through a central venous catheter. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 : Schematic depiction of the structures of the lead compound tTF-NGR with an N-terminal His-tag for detection and purification, full-length tTF, and the targeting peptide with a C-terminal NGR portion.

[0023] Figure 2: Depicts the targeting and accumulation of TF activity to tumor vessels, and the induction of tumor vascular thrombosis and infarction leading to tumor cell death. A, Components of tumor vascular infarction at the start of treatment; B, FXa, factor X activation (light blue), FVIIa, factor VII activation (blue) during tumor vascular coagulation.

[0024] Figure 3 : The therapeutic activity and mode of action of tTF-NGR as assessed by different imaging techniques are described. 1. Tumors appear blue (A. Left: tTF-NGR; A. Right: vehicle control). Macroscopic congestion and vascular rupture are visible several hours after tTF-NGR injection. 2. Tumor intravascular enhanced magnetic resonance imaging (MRI) shows the anatomy of the tumor (A, B) and the dramatic drop in blood flow from high blood flow (C) to low blood flow (D) within a few hours after tTF-NGR application. The vascular volume fraction was quantified to decrease by >1 log. 3. H&E staining of sarcoma xenograft tissue (A, thrombosis, congestion, and vascular rupture after tTF-NGR; B, saline control, normal vasculature). 4. In vivo fluorescence reflectance imaging of vascular anatomy after tTF-NGR by intravascular fluorescent fibrin (B). A, before tTF-NGR; B, 1 hour after tTF-NGR; C, vascular rupture 24 hours after tTF-NGR.

[0025] Figure 4: Depicting the synergistic activity of tTF-NGR and Trabectedin in human vascular endothelial cells (HUVEC). A, An example of a flow cytometry experiment showing PS upregulation by Trabectedin on the HUVEC cell membrane. PS was 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 h incubation). B, Seven different experiments with different doses of trabectedin were evaluated, with increasing doses of trabectedin inducing PS upregulation in HUVECs after 8 and 12 h (P values ​​< 0.0001 for all time points when compared to non-trabectedin control (NTC). Propidiumiodide was used as an internal necrosis control. C, Trabectedin-dependent increase in the procoagulant activity of HUVECs after binding to tTF-NGR (black, without trabectedin, gray, with trabectedin (10 nM, 8 h)), and this effect was completely abolished by masking PS with different doses of Annexin V. Mean + standard error and p values ​​for at least 4 analyses in 3 experiments each.

[0026] Figure 5: Depicting the synergistic activity of tTF-NGR and Trabectedin in HT1080 human sarcoma cells. A, PS upregulation was significant on HT1080 sarcoma cells compared to the no-trabectedin control (black) for 6 different experiments evaluated with 10 nM of trabectedin (grey) and an incubation time of 8 h. Mean + SE (p-value = 0.001). Propidium iodide was used as an internal necrosis control. B, Upon combination with tTF-NGR (black, without trabectedin, grey, with trabectedin (10 nM, 8 h)), the procoagulant activity of HT1080 sarcoma was dependent on trabectedin, and this effect was completely abolished by masking of PS with different doses of annexin V. Mean + SE and p-values ​​for 4 experiments. Figure 6 : In vivo therapeutic results of trabectedin combined 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 plus tTF-NGR (5 hours after trabectedin), with the same dose and schedule (Control n=8, tTF-NGR n=9, Trabectedin n=9, Combination n=11). DETAILED DESCRIPTION

[0027] Antivascular compounds in the form of bifunctional molecules, which have one conformational moiety targeting a specific structure on TECs and have an antitumor payload as a second conformational moiety, are known in the prior art. Such vascular targeting compounds are intended to target multiple tumor types through common tumor vasculature. Drugs targeting tumor cells containing only one histological entity have been approved for the treatment of specific tumors, such as moABs targeting tumor cell-associated molecules carrying toxins (brentuximab (CD30+ Hodgkin's disease), emtansine (HER2+ breast cancer)).

[0028] Another class of fusion proteins is named tTF-NGR, in which the nonspecific membrane anchor of tissue factor (TF) is replaced by the NGR motif binding CD13. CD13 is an aminopeptidase that occurs selectively on stimulated and growing ECs, as on TECs, and is expressed at low levels in other tissues (http: / / www.proteinatlas.org / ENSG00000166825-ANPEP / tissue). In some normal tissues (such as small bile ducts), CD13 expression further exists, but does not interfere with the application of CD13 targeting TF, because this molecule is only active in locations with strong coagulation ability (such as blood vessels) and is inactive elsewhere. Using this fusion protein ( Figure 1 ), tTF-activity is targeted and accumulated in tumor vessels, inducing tumor vascular thrombosis and infarction, leading to tumor cell death ( Figure 2 ).

[0029] These fusion proteins were tested in vitro and in vivo for basic therapeutic properties such as procoagulant activity, specific binding to the respective target molecules on stimulated endothelial cells (EC) or pericytes, in vivo intratumoral accumulation, in vivo intratumoral coagulation activation, tumor vasculature occlusion and blood flow inhibition, pharmacodynamic properties including therapeutic antitumor activity in human tumor xenografts from different tissue sources, and finally safety and toxicology in rodents and non-rodents. The results showed that while maintaining intact procoagulant activity, tTF-NGR specifically bound CD13 on growing ECs, causing tumor vasculature occlusion and infarction, leading to 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). No resistance was observed after multiple treatments.

[0030] In addition to the unique advantages of tTF-NGR, the present invention unexpectedly found that tTF-NGR and Trabectedin can show synergistic therapeutic activity when used in combination. Trabectedin is an anti-tumor drug originally isolated from a sea squirt, Ecteinascidia turbinata. This drug exerts its anti-tumor activity by binding to the minor groove of DNA during replication, resulting in double-strand breaks in the double helix. In addition, Trabectedin has been found to have a multi-effect mechanism in regulating inflammatory mediators in the tumor microenvironment. This effect may be achieved by selectively inhibiting the production of pro-inflammatory cytokines and chemokines, such as interleukin-6 (IL-6), chemokine ligand 2 (CCL2), matrix binding protein pentanelin 3 (PTX3) and vascular endothelial growth factor (VEGF). In addition, Trabectedin consumes macrophages in tumor tissues, and macrophage targeting appears to be a key component of its anti-tumor activity. Trabectedin as a single agent is often used as a standard second-line treatment for metastatic or refractory STS. As shown in the present invention, in the combined use of tTF-NGR and trabectedin, the two drugs showed higher anti-sarcoma activity in xenograft models compared with standard trabectedin monotherapy or tTF-NGR monotherapy. In view of the multiple modes of action of trabectedin on tumor cells and different cellular and molecular components of the tumor matrix, the combined therapeutic effect of trabectedin and tTF-NGR is unpredictable. tTF-NGR showed preclinical activity in human STS xenografts with tumor vascular occlusion and infarction, and the anti-tumor activity of trabectedin in these models can be improved in vivo by encapsulating trabectedin in the tumor, thereby obtaining stronger anti-tumor activity and longer duration. Conversely, trabectedin increases the procoagulant effect of tTF-NGR in tumor blood vessels. The binding side CD13 of tTF-NGR is strongly expressed on the vascular system and / or tumor cells of human STS. This provides a biomarker for patient selection for the combined treatment, and the predictability of the combined treatment activity can be further studied.

[0031] The present invention further provides a clinical dosage regimen for the combined treatment of cancer patients, particularly soft tissue sarcomas, using tTF-NGR and trabectedin. The present invention provides a composition containing trabectedin for treating cancer in an individual, wherein the treatment comprises the following steps:

[0032] (a) administering to the individual an effective amount of a composition comprising trabectedin, and

[0033] (b) then administering to the individual an effective amount of a composition comprising a tTF-NGR protein (preferably comprising or having SEQ ID NO: 2). The present invention also provides a composition comprising a tTF-NGR protein for treating cancer in an individual, wherein the treatment comprises the following steps:

[0034] (a) administering to the individual an effective amount of a composition comprising trabectedin, and

[0035] (b) subsequently administering to the individual an effective amount of a composition comprising a tTF-NGR protein (preferably comprising or having SEQ ID NO: 2).

[0036] In another embodiment, the composition comprising trabectedin and the composition comprising tTF-NGR (preferably comprising or having SEQ ID NO: 2) are administered to the individual by intravenous infusion.

[0037] In one embodiment, the composition comprising trabectedin is administered to the individual by 24-hour intravenous infusion. The dose of trabectedin is preferably 1.5 mg / m 2 .

[0038] In one embodiment, the tTF-NGR protein has the sequence of SEQ ID NO:2.

[0039] The first dose of tTF-NGR protein can be administered within 1 minute to 1 hour after the end of the administration of trabectedin. Preferably, the tTF-NGR protein is administered to the individual by 1 hour intravenous infusion. More preferably, after the administration of trabectedin, the tTF-NGR protein is repeated once a day for 4 consecutive days, for example: trabectedin at 8 am on Monday to 8 am on Tuesday, and then tTF-NGR is administered no later than 9 am on Tuesday, and then on the following days (the last tTF-NGR is on Friday).

[0040] In another embodiment, tTF-NGR protein is administered at 3 mg / m 2 Preferably, the tTF-NGR protein is infused in a 0.9% NaCl solution, with a total infusion volume of 100 ml. More preferably, the tTF-NGR protein is infused through a central venous catheter.

[0041] Therefore, in a preferred embodiment, 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:

[0042] (a) administering to the individual an effective amount of a composition comprising trabectedin, and

[0043] (b) subsequently administering to the individual an effective amount of a composition comprising the tTF-NGR protein of SEQ ID NO: 2; and

[0044] wherein trabectedin is administered to the individual by 24-hour intravenous infusion; and

[0045] The first administration of tTF-NGR protein begins between 1 minute and 1 hour after the end of trabectedin administration; and

[0046] wherein the tTF-NGR protein is administered to the individual by 1 hour intravenous infusion; and

[0047] The tTF-NGR protein was administered once a day for 4 consecutive days after the administration of trabectedin.

[0048] Preferably, the treatment cycle is 3 weeks. Patients receive repeated treatment until disease progression (iRECIST; Seymour L, Lancet Oncol. 2017), in the absence of other discontinuation criteria, as long as neither the patient nor the investigator requires discontinuation of treatment.

[0049] In one embodiment, the cancer in the individual is inoperable, metastatic or refractory. In a further embodiment, the cancer is a soft tissue sarcoma, and is preferably selected from the group consisting of dedifferentiated liposarcoma, myxoid liposarcoma, pleomorphic liposarcoma, adult fibrosarcoma, myxofibrosarcoma, leiomyosarcoma, rhabdomyosarcoma, angiosarcoma, synovial sarcoma and undifferentiated sarcoma.

[0050] For the purposes of this application, the following terms shall have the meanings used below and in the claims, unless otherwise stated.

[0051] "Patients" refers to mammals, preferably humans. In some embodiments, the patients are those who have failed standard first-line treatment or are not eligible for standard treatment.

[0052] "Therapeutically effective amount" refers to the amount of a compound that, when administered to a subject to treat a disease state, is sufficient to produce an improvement in the disease state.

[0053] "Trabectedin" refers to a tetrahydroisoquinoline alkaloid CAS (Chemical Abstracts Service) registration number 0114899-77-3, as described in the literature (eg Germano et al., Cancer Cell. 2013; 23(2): 249-262) and available from Pharma Mar, Madrid, Spain

[0054] "Treatment" or "treating" refers to the administration of any therapeutically effective amount of a compound, including:

[0055] inhibiting a disease in a human who is experiencing or displaying pathology or symptoms of the disease (i.e., preventing further development of the pathology and / or symptoms), or

[0056] - Ameliorating a disease in a human who is experiencing or displaying disease pathology or symptoms (ie, reversing the pathology and / or symptoms).

[0057] "iRECIST" refers to a series of modifications to the Response Evaluation Criteria in Solid Tumours (RECIST version 1.1) that account for apparent tumor growth that sometimes occurs before the tumor burden begins to decline, a phenomenon known as pseudoprogression. These responses occur in a small but corresponding fraction of patients receiving checkpoint inhibitors and other immunomodulators. The iRECIST guidelines (Seymour L, Lancet Oncol. 2017) were developed by a team led by Lesley Seymour, MD, PhD, an oncologist with the Canadian Cancer Trials Group at Queen's University in Kingston, Ontario.

[0058] "PFS" refers to progression-free survival, which was evaluated according to iRECIST at week 15 (i.e., after 5 cycles), then at weeks 24, 33, 42, and 51 (i.e., every 9 weeks or after 3 cycles adjusted to a 3-week cycle length), and every 3 months thereafter. The reason for using the iRECIST modification of RECIST for evaluation was that it was observed in preclinical studies and clinical cases that intratumoral congestion and swelling and vascular destruction could occur after tTF-NGR application, leading to pseudoprogression.

[0059] “OS” refers to the 12-month and 18-month overall survival rates.

[0060] "CR" means complete response, i.e., disappearance of all target lesions. The short axis of any pathological lymph node (whether target or non-target) must be reduced to 10 mm.

[0061] "PR" refers to partial response, i.e., a reduction of at least 30% in the sum of target lesion diameters, with the sum of baseline diameters as a reference.

[0062] "PD" means progressive disease, which means that the sum of the diameters of the target lesions has increased by at least 20%, taking the smallest sum on study as reference (including the baseline sum if smallest on study). 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 the rules described in Chapter 6.1.1 must be followed before a patient's progression can be used to end treatment.)

[0063] “SD” refers to stable disease, which means that neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as PD was observed, using the smallest summed diameter as a reference in the study.

[0064] "TF" refers to tissue factor, a protein known as tissue factor as described in the prior art, for example, in Huang et al., Science, 1997.

[0065] "tTF" refers to amino acids 33 to 251 of the tissue factor protein known as the truncated active form, also described in Huang et al., Science, 1997. The tTF sequence is comprised of the fusion protein sequence of SEQ ID NOs: 1 and 2 (amino acids 47 to 264 of SEQ ID NO: 2).

[0066] Example 1. Improving the therapeutic activity of tTF-NGR and trabectedin

[0067] The scientific hypothesis for combining tTF-NGR with trabectedin is that the pro-apoptotic activity of trabectedin on human vascular endothelial cells (HUVEC) and tumor cells significantly increases the presence of phosphatidylserine (PS) in the outer leaflet of the phospholipid bilayer that constructs the cell membrane (see Figure 4A and B, taking HUVEC as an example), by optimizing the phospholipid environment in the outer cell membrane, the procoagulant effect of tTF NGR in the tTF NGR: Factor VIIa: Factor X complex on the cell membrane was enhanced ( Figure 4C ). This effect was specifically dependent on the presence of PS, as it could be completely abolished by masking PS by preincubation with Annexin V.

[0068] Since part of the inner vascular cell layer of the newly formed blood vessels in tumor tissue is not formed by endothelial cells but by tumor cells, which is called "vascular mimicry", HT1080 human sarcoma cells were used instead of HUVECs for vascular simulation experiments (see Figure 5).

[0069] To improve activity in both directions, a pharmacokinetic approach was used to study the combination of trabectedin and tTF NGR, with tTF NGL administered approximately 5 hours after trabectedin injection. The results showed a significant improvement in therapeutic activity compared to either drug alone in the human STS xenograft model HT1080 ( Figure 6 ).

[0070] Example 2. tTF-NGR Phase I Clinical Study

[0071] tTF-NGR is produced in E. coli using a clinical-grade manufacturing process and a 4-step HPLC purification. The manufacturing process and GMP facilities are authorized by the regional government (Manufacturer's Authorisation by ) and the Federal Paul Ehrlich-Institute (PEI). Toxicity evaluations have been completed in mice, rats, guinea pigs and dogs according to EU-guidelines S6 and S9. The limiting toxicity in the most sensitive species (mouse) was pulmonary embolism, and the therapeutic safety window of tTF-NGR in this species was 1:4 (therapeutic dose: LD10 dose = 1:5).

[0072] An overview of the Phase I clinical study of tTF-NGR is shown in Table 1.

[0073] Table 1: Phase I clinical studies conducted during tTF-NGR progression

[0074]

[0075] In a phase I trial (EudraCT-No.: 2016-003042-85; NCT02902237) in patients with advanced cancer, tTF-NGR was dissolved in 0.9% NaCl and infused over 1 hour via a central venous catheter. This trial was the first of its kind, with repeated contrast-enhanced ultrasound (CEUS) and MRI as guidance to assess blood flow reduction in tumor signal lesions. The study design used a "5-day q day 22" schedule. At 4 mg / m 2 / d, a dose-limiting toxicity (DLT) was observed as a transient increase in troponin T hs without any clinical sequelae. This sensitive laboratory DLT was rapidly reversible and allows for early monitoring of subsequent dose adjustments in future trials. In addition, at 5 mg / m 2One case of CTCAE grade II deep vein thrombosis of the lower extremity was observed (1 patient, completely resolved) at 4 mg / m 2 Central venous catheter-related venous thrombosis (CTCAE grade II, resolved) was observed at 3 mg / m 2 Transient ischemic attack (CTCAE II, regression) was observed in one patient with left atrial angiosarcoma at this dose, which was plausibly related to tTF-NGR but not necessarily caused by tTF-NGR alone. Therefore, the trial was completed at the recommended phase II dose (RPIID) of 3 mg / m 2 / dx 5, qd 22. The study drug (IMP) was administered in 100 mL 0.9% NaCl via PORT central venous access as a rate-controlled infusion for 1 hour.

[0076] The pharmacokinetic studies in this study showed that the mean terminal half-life of tTF-NGR was 8.99 h. Since the level of tTF-NGR was always reached before the start of the next cycle, there was no accumulation during repeated cycles.

[0077] In patients with measurable CEUS and MRI, a specific inhibition of tumor blood flow (intratumoral blood circulation) was observed, without a reduction in blood flow to normal organs. Tumor blood flow inhibition was >1-log-step. In addition, in some metastases, rapidly evolving areas were observed with MRI, interpreted as areas of intratumoral hemorrhage and necrosis, similar to observations in xenograft models. No CR or PR has been observed in any of the patients treated to date. However, two patients had stable disease (SD) several months after treatment. Human anti-fusion protein antibodies (HAFA) were detected in a minority of patients without clinical symptoms (allergic or anaphylactoid reactions) or neutralizing capacity when associated with tumor blood flow inhibition on MRI or CEUS.

[0078] In summary, tTF-NGR can be safely used, and the phase I observations prove the principle of clinical tumor blood flow inhibition with a good therapeutic range. In addition, there are multiple effective methods to counter the theoretical systemic toxicity (heparin, COX inhibitors, dual platelet inhibition with aspirin and P2Y12 inhibitors, fibrinolysis).

[0079] Example 3. Clinical study design to detect the anti-sarcoma activity of tTF-NGR combined with trabectedin.

[0080] 3.1 Study design

[0081] The study design included a phase II / III, open-label, randomized, controlled study of subjects with metastatic or refractory soft tissue sarcoma with a total enrollment duration of 36 months.

[0082] Participants in this study were patients aged 18-75 years with advanced or metastatic soft tissue sarcoma after failure of first-line anthracycline-containing therapy or contraindications to these drugs. Patients had to have histological 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 type, pleomorphic), angiosarcoma, synovial sarcoma, undifferentiated sarcoma. CD13 positivity (grade 1+; Kessler T et al. Translational Oncology 2018) on central histology was a prerequisite for study entry. Participants had to 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) criteria 1.1. The lesion should not have been irradiated during the previous treatment. Participants were also required to have a life expectancy of at least 3 months, ECOG ≤ 2, and no conflict for trabectedin. 120 evaluable patients were included and assigned in parallel in a 1:1 manner to one of two different groups as described below. Randomization was based on a composite score of CD13 1+ / 2+ vs CD13 3+.

[0083] This study is divided into two parts, which are carried out in sequence:

[0084] Phase II:

[0085] Prior to the randomized Phase III portion of the study, there was a Phase II safety cohort of 6 patients, each receiving at least 3 cycles of the combination outlined in Arm 2 (see below, 1.5mg / m2 Trabectedin + 3mg / m2 tTF NGR) to confirm the safety of the combination. If there was a dose-limiting toxicity (DLT) in this Phase II cohort, the dose modification schedule of tTFNGR was planned to be modified to 2mg / m 2 If there are further tolerability issues, then plan to increase the dose to 0.5 mg / m 2 The dose will be further reduced in the tTFNGR step. The safe dose will then be transferred to the randomized Phase III of the study. The final dose of tTF NGR determined to be safe in this combination must be applied to 6 patients for 3 cycles each. After the DSMB judges the safety of the Phase II cohort, the randomized (Phase III) part of the study will be opened.

[0086] Phase III:

[0087] In the Phase III part of the study, 120 patients were randomized 1:1 into two groups. The primary objective was to evaluate the efficacy of tTF-NGR combined with trabectedin, measured by the median progression-free survival in the combination therapy group (Arm 2) versus the trabectedin monotherapy group (Arm 1).

[0088] ARM 1: Patients received 1.5 mg / m 2 Trabectedin is administered as a 24-hour central venous (iv) infusion on d 1, qd 22x until disease progression or further use is contraindicated (pre-medication according to institutional guidelines: e.g., 20 mg dexamethasone).

[0089] ARM 2: As in Arm 1, patients received standard trabectedin plus 3 mg / m2 / day for 4 consecutive days after each trabectedin cycle. 2 Dosing of tTF-NGR (1-hour rate-controlled infusion, PORT central venous access, 100 mL NaCl) (interval between end of trabectedin infusion and tTF NGR <1 hour): e.g., trabectedin Monday 8 AM to Tuesday 8 AM, then tTF-NGR Tuesday 9 AM and subsequent days (last tTF-NGL on Friday), qd 22x until disease progression or further use is contraindicated.

[0090] As study outcomes were assessed on an intention-to-treat basis, all randomized patients were part of the efficacy population assessed by central iRECIST at study completion.

[0091] Treatment in both arms was given as an outpatient. Some patients may require hospitalization for a 24-hour infusion of trabectedin. All patients received best supportive care (BSC) according to institutional guidelines.

[0092] Anticancer activity was assessed clinically at week 9, clinically and radiologically at week 15, and then every 9 weeks (adjusted to a cycle length of 3 weeks) until week 51 and every 3 months thereafter. Clinical decisions on the application of the next cycle were made at week 9. Radiological examinations and clinical-based decisions were performed starting at week 15. This process 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 cycle length of 3 weeks), and mOS, OS rate, and ORR at 12 and 18 months were calculated at 3-month intervals thereafter.

[0093] 3.2 Safety evaluation

[0094] Safety assessments were performed continuously during study participation and included standard laboratory evaluations. The incidence of AEs was summarized by severity in all patients who took at least one study drug.

[0095] Patients received repeated treatment until one of the following criteria was met (in the absence of any other withdrawal criteria):

[0096] 1) Unacceptable toxicity prevents further treatment;

[0097] 2) disease progression (as described below);

[0098] 3) The patient or investigator requests to stop treatment.

[0099] Patients who discontinue study treatment in the absence of disease progression should not receive further cancer treatment until disease progression unless there is a clear reason to continue with an alternative therapy.

[0100] Application of the next treatment cycle may be postponed up to 21 days due to the following reasons:

[0101] 1) Due to the patient's clinical condition, or

[0102] 2) The decision will be made by the researcher after discussion with the chief researcher, Professor Schliemann.

[0103] After the start of treatment, detailed follow-up was performed at weeks 9 and 15, then every 9 weeks (adjusted to a 3-week cycle length) until week 51, and every 3 months thereafter.

[0104] 3.3 Evaluation of the anticancer activity of the combination of tTF-NGR and trabectedin

[0105] This study used a modification of iRECIST to determine progression and PFS, similar to the study of checkpoint inhibitors (Seymour L, Lancet Oncol. 2017). After the study was completed, the final iRECIST judgment and comparison of PFS between the two groups were performed in a blinded manner by an independent imaging core laboratory at the Dept. of Radiology, LMU, Munich (Prof. M. Wildgruber). The iRECIST interim judgment of individual patient safety EOT was completed by local investigators at the local research center.

[0106] Anticancer activity (iRECIST modified) was assessed (clinically and radiologically) at weeks 9 and 15, with follow-up every 9 weeks (adjusted to a cycle length of 3 weeks) until week 51 and every 3 months thereafter.

[0107] Overall response assessment is similar to RECIST criteria version vs 1.1:

[0108] Complete Response (CR): All target lesions disappear. The short axis of any pathological lymph node (whether target or non-target) must be reduced to <10mm.

[0109] Partial Response (PR): The sum of target lesion diameters decreased by at least 30%, using the sum of baseline diameters as a reference.

[0110] Progressive Disease (PD): The sum of target lesion diameters increases by at least 20%, with the smallest sum on study as reference (including the baseline sum if smallest on study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm.

[0111] Stable Disease (SD): using the smallest summed diameter as reference in the study, with neither sufficient shrinkage to qualify as PR nor sufficient increase to qualify as PD.

[0112] Lymph nodes identified as target lesions should always have their actual short axis measurements recorded (measured in the same anatomical plane as the baseline examination), even if the node retracts to less than 10 mm during the study. This means that when a lymph node is listed as a target lesion, the “sum” of lesions may not be zero even if CR criteria are met, because normal lymph nodes are defined as having a short axis <10 mm. Therefore, the CRF or other data collection method can be designed to record target lymph node lesions in a separate section, and each node must achieve a short axis <10 mm to qualify for CR. For PR, SD, and PD, the actual short axis measurement of the node will be included in the sum of target lesions.

[0113] 3.4 Evaluation of median progression-free survival (mPFS) and progression-free survival (PFS) rate The PFS time of all randomized subjects was evaluated. The duration was defined as from the start of randomization to progression or death from any cause. The median PFS (mPFS) was calculated. The PFS rate according to iRECIST was evaluated at week 15 (after 5 cycles) and then at weeks 24, 33, 42, and 51 (i.e., every 9 weeks or after 3 cycles adjusted to a cycle length of 3 weeks) and at intervals of 3 months.

[0114] 3.5 Evaluation of Median Overall Survival (mOS) and Overall Survival (OS) Rate

[0115] All randomized patients were evaluated for OS duration. Duration was defined as the period from randomization to death from any cause. Median OS duration and OS rate were calculated at 12 and 18 months. Thereafter, survival of patients was monitored at regular follow-up for 3 months.

[0116] References:

[0117] Denekamp and Hobson, Endothelial-Cell proliferation in experimental tumors, Br. J. Cancer (1982) 46,711.

[0118] 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 trabectedinin advanced soft tissue sarcoma: beyond lipo-and leiomyosarcoma. Drug Design, Development. and Therapy 9:5785-5791,2015.

[0119] Kessler T,Baumeier A,Brand C,Grau M,Angenendt L,Harrach S,Stalmann U,Schmidt LH,Gosheger G,Hardes J,Andreou D,Dreischalück J,Lenz G,Wardelmann E,Mesters RM, 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.

[0120] 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.

[0121] 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.

[0122] 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. SEQUENCE LISTING <110> Anturik Pharmaceutical Co., Ltd. <120> Trabectedin for the Treatment of Cancer <130> P 109842 <160> 2 <170> BiSSAP 1.3.6 <210> 1 <211> 225 <212> PRT <213> Artificial Sequence <220> <223> tTF-NGR fusion protein <400> 1 Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln 20 25 30 Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val 50 55 60 Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala 65 70 75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr 100 105 110 Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val 180 185 190 Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Gly Asn Gly Arg Ala His 210 215 220 Ala 225 <210> 2 <211> 271 <212> PRT <213> Artificial Sequence <220> <223> His-tTF-NGR fusion protein <400> 2 Met His His His His His His Ser Ser Gly Leu Val Pro Arg Gly Ser 1 5 10 15 Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp 20 25 30 Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met Gly Ser Gly 35 40 45 Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn 50 55 60 Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr 65 70 75 80 Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe 85 90 95 Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp 100 105 110 Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn 115 120 125 Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro 130 135 140 Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln 145 150 155 160 Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu 165 170 175 Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val 180 185 190 Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser 195 200 205 Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp 210 215 220 Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro 225 230 235 240 Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met 245 250 255 Gly Gln Glu Lys Gly Glu Phe Arg Gly Asn Gly Arg Ala His Ala 260 265 270

Claims

1. A composition comprising an effective amount of trabectedin for administration to an individual and an effective amount of tTF-NGR protein for subsequent administration to the individual, thereby treating cancer in the individual.

2. The composition of claim 1, wherein the cancer is inoperable, metastatic or refractory.

3. The composition of claim 1, wherein the cancer is soft tissue sarcoma.

4. The composition of claim 3, 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.

5. The composition according to any one of claims 1 to 4, wherein trabectedin and tTF-NGR protein are administered to the individual by intravenous infusion.

6. The composition according to any one of claims 1-4, wherein trabectedin is administered to the individual by 24-hour intravenous infusion.

7. The composition according to any one of claims 1 to 4, wherein trabectedin is administered at 1.5 mg / m 2 dosage administration.

8. The composition according to any one of claims 1 to 4, wherein the tTF-NGR protein comprises or has the sequence of SEQ ID NO:

2.

9. The composition according to any one of claims 1-4, wherein the first administration of the tTF-NGR protein is started 1 minute to 1 hour after the end of the administration of trabectedin.

10. The composition of any one of claims 1-4, wherein the tTF-NGR protein is administered to the individual by 1 hour intravenous infusion.

11. The composition according to any one of claims 1 to 4, wherein the administration of the tTF-NGR protein is repeated once a day for the next 4 consecutive days.

12. The composition according to any one of claims 1 to 4, wherein the tTF-NGR protein is administered at 3 mg / m 2 / d dose administration.

13. The composition according to any one of claims 1 to 4, wherein the tTF-NGR protein is administered in a 0.9% NaCl solution with a total infusion volume of 100 ml.

14. The composition of any one of claims 1-4, wherein the tTF-NGR protein is infused via a central venous catheter.

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