EA049199B1 - COMPOSITION CONTAINING tTF-NGR FOR USE IN THE TREATMENT OF SOFT TISSUE SARCOMA - Google Patents

Abstract

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

Description

Field of invention

The invention relates to the use of tTF-NGR and trabectedin for the treatment of cancer, in particular soft tissue sarcoma. The inventors have unexpectedly found that the combination of tTF-NGR and trabectedin provides improved, preferably synergistic, therapeutic activity. Without being bound by theory, the improvement may be due to several effects. Vascular tumor occlusion with tTF-NGR prolongs intratumoral accumulation of trabectedin, which leads to higher antitumor efficacy. In addition, 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, which leads to higher phosphatidylserine (PS) levels in these cells.

Prerequisites for the creation of an invention

Soft tissue sarcomas (STS) are a rare group of heterogeneous mesenchymal tumours originating from connective tissue. STS consists of over 100 different subtypes, which collectively account for 1% of all adult cancers, and the incidence of STS in sarcoma in Europe ranges between 2-5/100,000 per year. They occur anywhere in the body, with common sites being the extremities, trunk, retroperitoneum and head/neck. Accurate histopathological diagnosis and classification, as well as tumor staging using imaging, are important as they influence multidisciplinary therapy, which is best performed in experienced reference centres. Due to the heterogeneity and rarity of the disease, diagnosis is often late.

Surgery is the first-line treatment for early-stage STS 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 the therapeutic approach is multidisciplinary. In cases with metastatic disease, the therapeutic goal is often limited to palliation and/or prolongation of progression-free survival rather than cure. First-line systemic therapy is based on doxorubicin monotherapy or in some combinations such as doxorubicin and ifosfamide. The combination of gemcitabine and docetaxel appears to be as effective as doxorubicin but causes more pronounced hematotoxicity and is sometimes used for second-line therapy. In addition to conventional chemotherapy, trabectedin, pazopanib and eribulin are alternatives for second- and subsequent-line therapy, and eribulin only for liposarcoma. However, overall survival of patients with advanced STS remains low in general. Thus, there is an unmet medical need for new therapeutic targets and agents for the treatment of this group of diseases.

Tumor growth and dissemination depend on intratumoral neovascularization to deliver nutrients and oxygen and remove waste products. Tumor endothelial cells (TECs) are essential for the construction of tumor vessels and, among other cell types such as fibroblasts and some myeloid and immune cells, belong to the tumor-supporting stroma. TECs express novel targets that are absent from the quiescent endothelial cells of the body's mature vasculature. Some of these TEC targets are clinically relevant for antiangiogenic cancer therapy, which has led to numerous drugs approved for cancer treatment (e.g., bevacizumab (anti-VEGF moAB), aflibercept (VEGF trap), ramucirumab (anti-VEGF-R moAB), sunitinib, sorafenib, pazopanib (examples of TEC receptor tyrosine kinase inhibitors)). The therapeutic activity of these drugs is limited to increasing survival by a few months, and the emergence of resistance limits the approach.

Conceptually distinct from antiangiogenic treatment is the antivascular approach. Antivascular drugs not only prevent vessel formation but also aim to destroy new vessels already present in tumors. Denekamp et al. proposed existing tumor vessels and tumor endothelial cells as target carriers for antivascular therapy (Denekamp et al., Br. J. Cancer, 1982). Tumor vasculature indeed represents therapeutic targets for antiangiogenic therapy, vessel destruction or vascular occlusion and thrombosis with subsequent tumor infarction. Huang et al. introduced the concept of tumor vessel occlusion using targeted tissue factor (TF) (Huang et al., Science, 1997). Pasqualini et al. characterized small peptides containing NGR (asparagine-glycine-arginine) binding 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 have prognostic value for patients with cancer of some, but not all, histologies examined (Tokuhara et al., Clin. Cancer Res., 2006).

Trabectedin is a tetrahydroisoquinoline alkaloid available as Yondelis® from Pharma Mar, Madrid, Spain. Trabectedin was first isolated from the Caribbean ascidian Ecteinascidia turbinata, but is now produced synthetically and is licensed in Europe and other countries for the treatment of adult patients with advanced soft tissue sarcoma (Ger

- 1 049199 mano et al., Cancer Cell. 2013; 23(2):249-262). Trabectedin acts as a DNA-binding agent, and binding to the DNA double helix results in interference with several transcription factors, DNA-binding proteins, and DNA repair pathways, resulting in G2/M cell cycle arrest and ultimately apoptosis. Furthermore, trabectedin has been found to provide a therapeutic option for metastatic lipo- and leiomyosarcoma, and is a promising candidate for the treatment of synovial sarcomas and high-grade undifferentiated pleomorphic sarcoma (De Santis et al., Drug Design, Develop, and Therapy 9: 5785-5791, 2015).

However, vascular-targeting thrombogenic agents may theoretically cause serious systemic side effects such as pulmonary embolism or stroke. Therefore, the development of safe and effective dosing regimens for the therapeutic use of vascular-targeting agents is of great preclinical and clinical importance.

The essence of the invention

The inventors unexpectedly found that the combination of tTF-NGR and trabectedin provides improved, preferably synergistic, therapeutic activity.

The invention relates to a composition containing trabectedin for use in the treatment of 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.

The invention also relates to a composition comprising the tTF-NGR protein for use in the treatment of 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.

In one embodiment of the invention, the cancer in the individual is inoperable, metastatic, or refractory.

In a further embodiment of the invention, 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.

In another embodiment of the invention, a composition comprising trabectedin and a composition comprising tTF-NGR are administered to an individual by intravenous infusion.

In another embodiment of the invention, the composition comprising trabectedin is administered to the individual via 24-hour intravenous infusion. Preferably, trabectedin is administered at a dose of 1.5 mg/ ^m2 .

In a preferred embodiment of the invention, the tTF-NGR protein comprises or has the sequence of SEQ ID NO: 2. In an alternative embodiment of the invention, 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 that comprises the 46 N-terminal amino acid residues. Although 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 NOs: 1 and 2 further comprise 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.

In another embodiment of the invention, the first administration of tTF-NGR protein is started between 1 minute and 1 hour after the end of the administration of trabectedin. Preferably, the tTF-NGR protein is administered to the individual via a 1-hour intravenous infusion. More preferably, the administration of tTF-NGR protein is repeated once a day for the next 4 consecutive days after the administration of trabectedin.

In a further embodiment of the invention, the tTF-NGR protein is administered at a dose of 3 mg/ ^m2 /day. Preferably, the tTF-NGR protein is administered in a 0.9% NaCl solution in a total infusion volume of 100 ml. More preferably, the tTF-NGR protein is administered through a central venous port.

Description of figures

Fig. 1: Schematic illustration of the structure of the tTF-NGR lead junction with an N-terminal His-tag for detection and purification of full-length tTF and a target peptide with an NGR motif at the C-terminus.

Fig. 2: Illustration of targeting and accumulation of TF activity in tumor vasculature, induction of tumor vessel thrombosis and infarction, leading to tumor cell death. A, Components of tumor vessel infarction at the start of therapy, and B, during tumor vessel clotting. FXa, factor X activated (blue), FVIIa, factor VII activated (blue).

Fig. 3: Illustration of the therapeutic activity and modes of action of tTF-NGR using different imaging techniques. 1. Blueish coloration of the tumor (A, left: tTF-NGR. A, right: vehicle control) several hours after tTF-NGR injection, macroscopically visualizing blood pooling and vascular disruption. 2. Contrast-enhanced intravascular magnetic resonance imaging (MRI) of the tumor showing the anatomy of the tumor (A, B) and the abrupt decrease in blood flow with high

- 2 049 199 high blood flow (C) to low blood flow (D) several hours after tTF-NGR application. The proportion of vascular volume was quantified as a >1 log decrease. 3. H&E staining of sarcoma xenograft tissue (A, thrombosis, blood pooling, and vascular disruption after tTF-NGR; B, saline control with normal vasculature). 4. In vivo fluorescence reflectance imaging showing vascular anatomy with intravascular fluorescent fibrin after tTF-NGR (B). A, before tTF-NGR; B, 1 h after tTF-NGR; C, vascular disruption 24 h after tTF-NGR.

Fig. 4: Illustration of the synergistic activity of tTF-NGR and trabectedin in human vascular endothelial cells (HUVEC). A, One example of a flow cytometry experiment showing the activation of PS by trabectedin on HUVEC cell membranes. PS is detected by the fluorescent PS-binding annexin V-FITC. PS-positive cells are shown in the right lower quadrant (LR) and increase from 7.21% without trabectedin to 29.22% with trabectedin (15 nM, 8 h incubation). B, Evaluation of 7 different experiments with different doses of trabectedin inducing PS activation in HUVEC at 8 and 12 h by increasing the dose of trabectedin (P values for all time points < 0.0001 compared to the no trabectedin control (NTC). Propidium iodide was used as a control for internal necrosis. C, Trabectedin-dependent increase in HUVEC procoagulant activity upon tTF-NGR binding (black, no trabectedin, gray, with trabectedin (10 nM, 8 h)) and complete abolition of this effect by PS masking with annexin V at different doses. Mean values of 3 experiments with at least 4-fold analysis in each + SE and p-values.

Fig. 5: Illustration of the synergistic activity of tTF-NGR and trabectedin in human sarcoma HT1080 cells. A, Evaluation of 6 different experiments with 10 nM trabectedin (gray) and 8 h incubation time showing significant PS activation on HT1080 sarcoma cells compared to the control without trabectedin (black). Mean + SE (p-value = 0.001). Propidium iodide was used as a control for internal necrosis. B, Trabectedin-dependent increase in the procoagulant activity of HT1080 sarcoma upon tTF-NGR binding (black, without trabectedin, gray, with trabectedin (10 nM, 8 h)) and complete abolition of this effect by PS masking with annexin V at different doses. Means of 4 experiments + SE and p-values.

Fig. 6: Therapeutic results of the combination of trabectedin with tTF-NGR in vivo in the HT1080 STS xenograft model. Control, PBS i.v.; tTF-NGR 1 mg/kg, day 0; trabectedin 0.1 mg/kg day 0; combination, trabectedin plus tTF-NGR (5 h after trabectedin) with identical doses and timing (control n=8, tTF-NGR n=9, trabectedin n=9, combination n=11).

Detailed description of the invention

Antivascular compounds in the form of bifunctional molecules targeting a specific TEC structure with one moiety and having an antitumor payload as a second moiety have been known in the prior art. This class of vascular-targeting compounds aims to target more than one tumor type via the common tumor vasculature. Drugs targeting tumor cells containing only one histological entity have been approved for the treatment of specific tumors, such as moABs to molecules associated with tumor cells carrying toxins (brentuximab vedotin (CD30+ Hodgkin's disease), trastuzumab emtansine (HER2+ breast cancer)).

Another class of fusion proteins, termed tTF-NGR, has been developed where the non-specific membrane anchor of tissue factor (TF) is replaced by a CD13-binding NGR motif. CD13 is an aminopeptidase selectively found on stimulated and growing EC as well as on TEC, with low expression in other tissues (http://www.proteinatlas.org/ENSG00000166825-ANPEP/tissue). In some normal tissues, such as small bile ducts, CD13 expression is also more than present, but does not interfere with the use of CD13-targeted TF, since this molecule is active only at the coagulation-competent site, such as blood vessels, and not elsewhere. With this fusion protein (Fig. 1), TF activity is targeted and accumulated in the tumor vasculature, and tumor vessel thrombosis and infarction are induced, leading to tumor cell death (Fig. 2).

These fusion proteins were tested in vitro and in vivo for key therapeutic properties such as procoagulant activity, specific binding to the corresponding target molecules on stimulated endothelial cells (ECs) or pericytes, intratumoral accumulation in vivo, intratumoral activation of coagulation in vivo, tumor vascular occlusion and blood flow inhibition, pharmacodynamic properties including therapeutic antitumor activity in human tumor xenografts of different histological origins, and finally safety in rodents and non-rodent animals and toxicology. As demonstrated by the experimental results, while maintaining full procoagulant activity, tTF-NGR specifically binds to CD13 on growing ECs, resulting in tumor vessel occlusion and infarction followed by tumor growth inhibition and regression (Fig. 3). The preclinical therapeutic activity of tTF-NGR was independent of tumor histology (e.g. melanoma, lung, breast, sarcoma, glioblastoma). Repeated treatment cycles did not reveal the development of resistance.

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In addition to the specific advantages of tTF-NGR, the present inventors have unexpectedly found that the combination of tTF-NGR and trabectedin can exhibit synergistic therapeutic activity. Trabectedin is an antitumor drug originally isolated from Ecteinascidia turbinata, a sea squirt. The drug exerts its antitumor activity by binding to the minor groove of DNA during replication, causing double-strand breaks in the double helix. Moreover, trabectedin has been found to have a pleiotropic mechanism of action in regulating inflammatory mediators in the tumor microenvironment. This effect is possibly achieved by selectively inhibiting the production of proinflammatory cytokines and chemokines, such as interleukin-6 (IL-6), chemokine ligand 2 (CCL2), matrix-binding protein pentraxin 3 (PTX3), and vascular endothelial growth factor (VEGF). In addition, trabectedin depletes macrophages in tumor tissue, and targeting macrophages appears to be a key component of its antitumor activity. Trabectedin as monotherapy is commonly used as standard 2nd-line therapy for metastatic or refractory STS. In combination of tTF-NGR with trabectedin, as shown in the present invention, both drugs demonstrated enhanced antisarcoma activity compared to standard trabectedin monotherapy or tTF-NGR monotherapy in a xenograft model. Given this multiple mechanism of action of trabectedin on tumor cells and various cells and molecular components of the tumor stroma, the combined therapeutic efficacy of trabectedin with tTF-NGR was unexpected. tTF-NGR has demonstrated preclinical activity in human STS xenografts in tumor vascular occlusion and infarction and may improve the antitumor activity of trabectedin in these in vivo models by trapping trabectedin within the tumor, resulting in more potent antitumor activity with a longer duration. Conversely, trabectedin enhances the procoagulant efficacy of tTF-NGR in the tumor vasculature. The binding site of tTF-NGR, CD13, is highly expressed in the vasculature and/or on human STS tumor cells. This provides a biomarker for patient selection for this combination that can be further studied to predict the activity of the combination treatment.

The invention further relates to clinical dosing regimens for the use of tTFNGR and trabectedin in combination therapy for the treatment of patients with cancer, in particular soft tissue sarcoma.

The invention relates to a composition comprising trabectedin for use in the treatment of 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 a tTF-NGR protein (preferably comprising or having SEQ ID NO: 2).

The present invention also relates to a composition comprising a tTF-NGR protein for use in the treatment of 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 a tTF-NGR protein (preferably comprising or having SEQ ID NO: 2).

In another embodiment of the invention, a composition comprising trabectedin and a composition comprising tTF-NGR (preferably comprising or having SEQ ID NO: 2) are administered to an individual via intravenous infusion.

In one embodiment of the invention, a composition comprising trabectedin is administered to an individual via 24-hour intravenous infusion. Preferably, trabectedin is administered at a dose of 1.5 mg/ ^m2 .

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

The first dose of tTF-NGR protein may be administered 1 minute and 1 hour after completion of trabectedin administration. Preferably, tTF-NGR protein is administered to the individual via a 1-hour intravenous infusion. More preferably, tTF-NGR protein administration is repeated once daily for the next 4 consecutive days after trabectedin administration, for example: trabectedin from 8:00 Monday to 8:00 Tuesday, then tTF-NGR on Tuesday no later than 9:00 AM and on subsequent days (last tTFNGR on Friday).

In a further embodiment of the invention, the tTF-NGR protein is administered at a dose of 3 mg/ ^m2 /day. Preferably, the tTF-NGR protein is administered in a 0.9% NaCl solution in a total infusion volume of 100 ml. More preferably, the tTF-NGR protein is administered through a central venous port.

In a preferred embodiment, the present invention thus relates to a composition comprising trabectedin or a tTF-NGR protein for use in the treatment of 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 a tTF-NGR protein having SEQ ID NO: 2; and

- 4 049199 wherein trabectedin is administered to the individual via a 24-hour intravenous infusion; and wherein the first administration of tTF-NGR protein is started between 1 minute and 1 hour after the end of administration of trabectedin; and wherein tTF-NGR protein is administered to the individual via a 1-hour intravenous infusion; and wherein administration of tTF-NGR protein is repeated once daily for the next 4 consecutive days after administration of trabectedin.

The preferred duration of a treatment cycle is 3 weeks. Patients are treated in repeated cycles until defined disease progression (iRECIST; Seymour L, Lancet Oncol. 2017) in the absence of other discontinuation criteria and until neither the patient nor the investigator requests treatment discontinuation.

In one embodiment of the invention, the cancer in the subject is inoperable, metastatic or refractory. In a further embodiment of the invention, 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.

Unless otherwise specified, for the purposes of this application, the following terms are intended to have the meanings set forth below as used in the description and claims.

The patients are mammals, preferably humans. In some embodiments, the patients are patients who have failed standard first-line treatment or who are not suitable for standard treatment.

A therapeutically effective amount means an amount of a compound that, when administered to a subject for the treatment of a disease state, is sufficient to effect an improvement in such disease state.

Trabectedin belongs to the tetrahydroisoquinoline alkaloid CAS registry number (Chemical Abstracts Service) 0114899-77-3, 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.

Treatment/cure means any administration of a therapeutically effective amount of a compound and includes inhibition of a disease in individuals experiencing or exhibiting the pathology or symptomatology of the disease (i.e., slowing the further progression of the pathology and/or symptomatology), or alleviation of the course of the disease in individuals experiencing or exhibiting the pathology or symptomatology of the disease (i.e., reversal of the pathology and/or symptomatology).

iRECIST refers to a series of modifications of the Response Evaluation Criteria in Solid Tumors (RECIST version 1.1) that take into account apparent tumor growth that sometimes occurs before tumor burden begins to decline, a phenomenon known as pseudoprogression. These reactions occur in a small but significant proportion of patients receiving checkpoint inhibitors and other immunomodulatory agents. The iRECIST guideline (Seymour L, Lancet Oncol. 2017) was developed by a team led by Leslie Seymour, MD, an oncologist at the Canadian Cancer Research Group, based at Queen’s University in Kingston, Ontario.

PFS refers to progression-free survival 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 for a 3-week cycle length), and then every 3 months. The reason for using the iRECIST modification of RECIST for assessment is the observation in preclinical studies and case reports that intratumoral edema may occur with tTF-NGR due to blood pooling and vascular rupture, leading to pseudoprogression.

OS refers to overall survival at 12 and 18 months.

CR refers to complete response, meaning the disappearance of all target lesions. Any abnormal lymph nodes (both target and non-target) must have a short-axis reduction of <10 mm.

PR refers to a partial response, which means a reduction in the sum of target lesion diameters by at least 30% using the baseline sum diameters as a reference.

PD refers to progressive disease, which means at least a 20% increase in the sum of the target lesion diameters using the lowest sum in the study as the reference (including the baseline sum if it is the lowest in the study). In addition to the 20% relative increase, the sum must also show an absolute increase of at least 5 mm. (Note: the appearance of one or more new lesions is also considered progression; the rules described in Chapter 6.1.1 must be followed before progression can be used to stop treatment of a patient.)

SD refers to stable disease, meaning that there is neither sufficient decrease to qualify PR nor sufficient increase to qualify PD, using the smallest sum diameters as the reference in the study.

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TF refers to tissue factor, a known tissue factor protein described in the prior art, such as Huang et al., Science, 1997.

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

Example 1. Improved therapeutic activity of tTF-NGR and trabectedin.

The scientific hypothesis of the combination of tTF-NGR and trabectedin is that the pro-apoptotic activity of trabectedin against human vascular endothelial cells (HUVEC) as well as tumor cells significantly increases the presence of phosphatidylserine (PS) on the outer leaflet of the phospholipid bilayer forming the cell membrane (see Fig. 4 A and B for HUVEC as an example), and due to this optimized phospholipid environment in the outer cell membrane, the procoagulant efficiency of tTF-NGR in the tTF-NGR:factor VΠa:factor X complex on the cell membrane is enhanced (Fig. 4 C). This effect is particularly dependent on the presence of PS, since it can be completely eliminated by masking PS by pre-incubation with annexin V.

Since in tumor tissues, part of the inner vascular cell layer of the new vascular network is formed not by endothelial cells, but also by tumor cells, which is called vascular mimicry, similar experiments were also carried out with human sarcoma cells HT1080 instead of HUVEC (see Fig. 5).

To exploit the improved activity in both directions, the combination of trabectedin with tTF-NGR was studied using a pharmacokinetic approach, administering tTF-NGR approximately 5 h after trabectedin administration. The results show a significant improvement in therapeutic activity compared to the two drugs alone in the human STS xenograft model HT1080 (Fig. 6). Example 2. tTF-NGR Phase I Clinical Study.

tTF-NGR was produced using E. coli by means of a clinical-grade manufacturing process and 4-step HPLC purification. The manufacturing process and the GMP facility are approved by the regional government (Regierungsprasidium manufacturer approval) and the Paul-Ehrlich-Institute (PEI). A toxicity assessment according to EU guidelines S6 and S9 was completed in mice, rats, guinea pigs and dogs. The limiting toxicity in the most sensitive species (mouse) was pulmonary embolism, in this species tTF-NGR has a therapeutic safety window of 1:4 (therapeutic dose:LD10 dose=1:5).

An overview of the phase I clinical study with tTF-NGR is presented in Table 1.

Table 1

Klin study Study Identification Number EudraCT-No: NCT02902237 Phase I study, conducted Study design/objectives 2016-003042-85 Phase I study of tTFNGR tTF-NGR via central venous access as daily 1-hour infusions for 5 days, every 22 days; intra-individual dose escalation was allowed in subsequent cycles. Patients were treated sequentially rather than in parallel during the dose escalation phase. Primary objective: To evaluate the maximum tolerated dose (MTD) and dose-limiting toxicity (DLT) of intravenous (IV) infusions of daily tTF-NGR applications for 5 days every 3 weeks in patients with relapsed or refractory cancer who have received all standard yue in progress p Population/ number of subjects Patients with solid tumors and lymphomas at advanced stages refractory to standard treatment. Number of subjects: 24 24 of which 17 were actually treated with tTF NGR >tTF developments Dosing regimens Initial dose: 1 mg/ ^m2 daily DLT: Dose-limiting toxicity (DLT) at 4 and 5 mg/ ^m2 daily Screening group: R.PIID screening group with 3.0 mg/ ^m2 tTF-NGR daily (x5) -NGR

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treatment known for their nosological entity before inclusion in the study. Secondary objectives: 1. To determine perfusion and vessel volume fraction of measurable tumor lesions compared to normal reference tissue before and after tTF-NGR administration using MRI and/or CEUS as a biological surrogate parameter for IMP biological activity in patients treated in the validation cohorts. 2. To obtain pharmacokinetic data.

A phase I clinical trial (EudraCT-No.: 2016-003042-85; NCT02902237) was conducted using tTF-NGR as a 1-hour infusion dissolved in 0.9% NaCl via a central venous line in patients with advanced cancer. This study was the first in its class to be guided by repeat contrast-enhanced ultrasound (CEUS) and MRI to assess blood flow reduction in tumor sentinel lesions. The study design utilized a 5-day every 22-day schedule.

At the 4 mg/ ^m2 /day dose, dose-limiting toxicity (DLT) was observed as transient troponin Ths levels increased without any clinical consequences. This sensitive laboratory DLT was rapidly reversible and allows for early monitoring for subsequent dose adjustment in future studies. In addition, one CTCAE grade II lower extremity deep vein thrombosis (in 1 patient, completely resolved) was also observed at the 5 mg/ ^m2 dose, a catheter-related central vein thrombosis was observed at the 4 mg/ ^m2 dose (CTCAE grade II and resolved), and a transient ischemic attack (CTCAE II resolved) in a patient with angiosarcoma in the left atrium was observed at the 3 mg/ ^m2 dose, events reasonably associated with, but not necessarily caused by, tTF-NGR alone. Thus, the study was completed with the recommended phase II dose (RPIID) of 3 mg/ ^m2 /day x 5, QD 22. The study drug (IMP) was administered via a central venous PORT access in 100 mL 0.9% NaCl as a controlled rate infusion over 1 hour.

Pharmacokinetic studies showed a mean terminal half-life of tTFNGR of 8.99 h. There was no accumulation with repeated cycles, as pre-treatment levels were always achieved before the start of the next cycle.

Patients showed specific inhibition of tumor blood flow (intratumoral blood flow) by CEUS and MRI without reduction of blood flow in normal organs. Inhibition of tumor blood flow reached >1 log step. Furthermore, some metastases on MRI showed rapid development of areas interpreted as intratumoral hemorrhage and areas of necrosis similar to those observed in xenograft models. None of the patients treated to date have shown CR or PR. However, two patients had stable disease (SD) for several months after treatment. Human antibodies to the fusion protein (HAFA) were detected in several patients without evidence of clinical symptoms (anaphylactic or anaphylactoid reactions) or neutralizing capacity in correlation with tumor blood flow inhibition on MRI or CEUS.

In conclusion, tTF-NGR can be safely administered and phase I observations provide proof of principle for tumor blood flow inhibition in the clinical setting and a promising therapeutic window. In addition, there are several effective methods to counteract the theoretical systemic toxicity (heparin, COX inhibitors, dual platelet inhibition with aspirin and P2Y12 inhibitors, fibrinolysis).

Example 3. Design of a clinical trial to test the antisarcoma activity of the combination of tTF-NGR and trabectedin.

3.1 Study design.

The study design consists of a phase II/III, open-label, randomized, controlled trial in subjects with metastatic or refractory soft tissue sarcoma for a total duration of 36 months.

Participants in the study are patients aged 18 to 75 years with widespread or

- 7 049199 metastatic soft tissue sarcoma after failure of first-line anthracycline-containing therapy or with contraindications to these drugs. Patients must have histological evidence of advanced unresectable or metastatic high-grade 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, undifferentiated sarcoma. CD13 positivity on central histology (grade 1+; Kessler T et al., Translational Oncology 2018) is a requirement for inclusion in the study. Participants must have at least one unidimensional lesion measurable by CT as defined by iRECIST criterion 1.1 (Response Evaluation Criteria in Solid Tumors; Seymour L, Lancet Oncol. 2017). This lesion must not have been irradiated in previous treatments. Participants are also required to have a life expectancy of at least 3 months with an ECOG <2 and no contraindications to trabectedin. 120 evaluable patients are enrolled and will be allocated in parallel in a 1:1 ratio to one of two different groups as outlined below. Randomization will be stratified by a composite score of CD13 1+/2+ versus CD13 3+.

The study is divided into 2 parts, which are carried out sequentially.

Part II of phase.

Prior to the randomised phase III portion of the study, there is a phase II safety cohort of 6 patients receiving at least 3 cycles of each of the combinations listed in cohort 2 (see below, 1.5 mg/ ^m2 trabectedin plus 3 mg/ ^m2 tTF-NGR) to confirm the safety of this combination. In case of dose-limiting toxicity (DLT) in this phase II cohort, a dose adjustment protocol for tTF-NGR to 2 mg/ ^m2 is planned, and in case of further tolerability issues, further de-escalation in 0.5 mg/ ^m2 tTF-NGR steps is planned. The safe dose is then carried forward to the randomised phase III portion of the study. The final tTF-NGR dose found to be safe in this combination is to be administered to 6 patients with 3 cycles each. The randomised (phase III) portion of the study will be opened following the safety assessment in the phase IIDSMB cohort.

Part III of phase.

In the phase III part of this study, 120 patients were randomized 1:1 into two different groups. The primary objective is to evaluate the efficacy of tTF-NGR in combination with trabectedin, measured as median progression-free survival in the combination treatment group (group 2) compared with trabectedin monotherapy (group 1).

Arm 1: Patients receive 1.5 mg/ ^m2 trabectedin as a 24-hour central intravenous (IV) infusion on day 1, QD 22 x until disease progression or contraindication to further use (premedication according to institutional guidelines: eg, 20 mg dexamethasone).

Arm 2: Patients receive standard trabectedin as per Arm 1 plus 3 mg/ ^m2 tTF-NGR (1-hour adjustable-rate infusion, PORT central venous access, 100 mL NaCl) daily for 4 consecutive days after each trabectedin cycle (<1 hour interval between end of trabectedin infusion and tTF-NGR: eg: trabectedin on Monday 8:00 AM to Tuesday 8:00 AM, then tTF-NGR on Tuesday 9:00 AM and thereafter (last tTF-NGR on Friday), QD 22x until disease progression or contraindication to further use.

Because the outcome assessment of the study is based on intention-to-treat, all patients after randomization are part of the efficacy population as assessed by the iRECIST central assessment after the end of the study.

Therapy in both groups can be performed on an outpatient basis. Some patients may require hospitalization for 24 hours of trabectedin infusion. All patients receive best supportive care (BSC) according to institutional guidelines.

Anticancer activity is assessed clinically at week 9 and clinically and by imaging at week 15 and then every 9 weeks (adjusted for a 3-week cycle length) until week 51 and every 3 months thereafter. The decision to administer the next cycle is made clinically at week 9. Imaging and a clinically based decision follow from week 15 onwards. This procedure is described in detail below. Median PFS, PFS (iRECIST), and DCR rates at 15, 24, 33, 42, and 51 (ie, every 9 weeks or after 3 cycles adjusted for a 3-week cycle length) and then at 3-month intervals, mOS, OS rates at 12 and 18 months, and ORR are calculated.

3.2 Safety assessment.

Safety assessments will be performed on an ongoing basis during study participation, including routine laboratory assessments. The incidence of AEs will be summarized by severity across all patients who received at least one dose of the study drug.

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Patients are treated in repeated cycles until one of the following criteria is met (in the absence of any other discontinuation criteria):

1) unacceptable toxicity precluding further therapy,

2) disease progression (as described below),

3) the patient or investigator requests that treatment be stopped.

Patients who discontinue study drug in the absence of disease progression should not receive further linear cancer therapy until disease progression unless there are clear reasons to continue alternative treatments.

The next cycle of therapy may be delayed for up to 21 days for the following reasons:

1) due to the clinical condition of the patient or

2) by decision of the researcher after discussion with the principal researcher, Prof. Schliemann.

Detailed follow-up visits after initiation of therapy are performed at weeks 9 and 15, then every 9 weeks (adjusted for a 3-week cycle length) until week 51, and then at 3-month intervals.

3.3 Evaluation of the anticancer activity of the combination of tTF-NGR and trabectedin.

The study uses a modification of iRECIST to define progression and PFS similar to the checkpoint inhibitor trials (Seymour L, Lancet Oncol. 2017). The final iRECIST assessment and comparison of PFS between both groups will be performed after study completion in a blinded manner in an independent core imaging laboratory at the Department of Radiology, LMU, Munich (Prof. M. Wildgruber). A dedicated iRECIST assessment to determine safe EOT for individual patients will be performed by local investigators at local study centers.

Anticancer activity (iRECIST modification) is 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 then every 3 months.

The overall response is assessed in a manner similar to the RECIST version 1.1 criteria:

Complete response (CR): Disappearance of all target lesions. Any abnormal lymph nodes (both target and non-target) should have a short-axis reduction of <10 mm.

Partial response (PR): reduction in the sum of target lesion diameters by at least 30% using baseline sum diameters as reference.

Progressive Disease (PD): An increase of at least 20% in the sum of the target lesion diameters using the smallest sum in the study as the reference (including the baseline sum if it is the smallest sum in the study). 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 decrease to qualify PR nor sufficient increase to qualify PD, using the smallest sum diameters as the reference in the study.

Lymph nodes identified as target lesions must always have an actual short axis measurement (measured in the same anatomical plane as the original study), even if the nodes regress to <10 mm in the study. This means that when lymph nodes are included as target lesions, the lesion sum may not be zero even if CR criteria are met, since a normal lymph node is defined as having a short axis <10 mm. Therefore, CRF or other data collection methods can be designed such that target nodal lesions are recorded in a separate section, where each node must achieve a short axis <10 mm to qualify as CR. For PR, SD, and PD, the actual short axis measurement of the nodes should be included in the target lesion sum.

3.4 Assessment of median progression-free survival (mPFS) and progression-free survival rates.

Time to PFS is assessed for all randomized subjects. Duration is defined as from randomization to progression or death from any cause. Median PFS (mPFS) is calculated. iRECIST PFS is assessed at week 15 (after 5 cycles), then at weeks 24, 33, 42, and 51 (ie, every 9 weeks or after 3 cycles adjusted for a 3-week cycle length), and then at 3-month intervals.

3.5 Assessment of median overall survival (mOS) and overall survival rates.

Survival (mOS) and overall survival.

The OS time is estimated for all randomized patients. The duration is defined from randomization to death from any cause. The median OS time and the OS rate at 12 and 18 months are calculated. Patient survival is then monitored at regular follow-up visits for 3 months.

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Claims (13)

Links 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, Develop, 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, Dreischaliick J, Lenz G, Wardelmann E, Mesters RM, Schwoppe C, Berdel WE*, Hartmann W*, Schliemann C*: AminopeptidaseN (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 CLAUSE OF INVENTION 1. Use of a composition comprising trabectedin for 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. 2. Use of a composition comprising a tTF-NGR protein for 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. 3. Use of a composition for the treatment of soft tissue sarcoma according to claim 1 or 2, wherein the cancer in the individual is inoperable, metastatic or refractory. 4. Use of a composition for treating soft tissue sarcoma according to any one of claims 1, 2, wherein the cancer is 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. 5. Use of a composition for treating soft tissue sarcoma according to any one of claims 1 to 4, wherein the composition containing trabectedin and the composition containing tTF-NGR are administered to the individual by intravenous infusion. 6. Use of a composition for treating soft tissue sarcoma according to any one of claims 1 to 5, wherein the composition containing trabectedin is administered to an individual by 24-hour intravenous infusion. 7. Use of a composition for the treatment of soft tissue sarcoma according to any one of claims 1-6, wherein trabectedin is administered at a dose of 1.5 mg/ ^m2 . 8. Use of a composition for treating soft tissue sarcoma according to any one of claims 1 to 7, wherein the tTFNGR protein comprises or has the sequence of SEQ ID NO: 2. 9. Use of a composition for the treatment of soft tissue sarcoma according to any one of claims 1 to 8, wherein the first administration of the tTF-NGR protein is started between 1 minute and 1 hour after the end of the administration of trabectedin. 10. Use of a composition for treating soft tissue sarcoma according to any one of claims 1 to 9, wherein the tTF-NGR protein is administered to an individual via a 1-hour intravenous infusion. 11. Use of a composition for treating soft tissue sarcoma according to any one of claims 1-10, wherein the administration of the tTF-NGR protein is repeated once a day for the next 4 consecutive days. 12. Use of a composition for treating soft tissue sarcoma according to any one of claims 1-11, wherein the tTF-NGR protein is administered at a dose of 3 mg/ ^m2 /day. 13. Use of a composition for treating soft tissue sarcoma according to any one of claims 1-12, wherein the tTF-NGR protein is administered in a 0.9% NaCl solution in a total infusion volume of 100 ml. -