WO2015058369A1

WO2015058369A1 - Use of aflibercept and docetaxel for the treatment of nasopharyngeal carcinoma

Info

Publication number: WO2015058369A1
Application number: PCT/CN2013/085764
Authority: WO
Inventors: Li Zhang; Yan Huang
Original assignee: Sanofi (China) Investment Co., Ltd.; Sanofi
Priority date: 2013-10-23
Filing date: 2013-10-23
Publication date: 2015-04-30
Also published as: WO2015059220A1

Abstract

The present invention concerns a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof, and a combined preparation or a composition comprising a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof and docetaxel, for use for treating patients with nasopharyngeal carcinoma. The invention also pertains to an article of manufacture comprising a packaging material, a polypeptide of SEQ ID NO: 1, or a biosimilar thereof, and a label or package insert contained within said packaging material indicating that the polypeptide or biosimilar thereof is indicated for patients with nasopharyngeal carcinoma. The invention further relates to a safe therapeutic dose of a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof, in combination with docetaxel, for use for treating patients with nasopharyngeal carcinoma. In one embodiment, the polypeptide is aflibercept.

Description

USE OF AFLIBERCEPT AND DOCETAXEL FOR THE TREATMENT OF

NASOPHARYNGEAL CARCINOMA

The present invention concerns a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof, and a combined preparation or a composition comprising a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof and docetaxel, for use for treating patients with nasopharyngeal carcinoma. The invention also pertains to an article of manufacture comprising a packaging material, a polypeptide of SEQ ID NO: 1 , or a biosimilar thereof, and a label or package insert contained within said packaging material indicating that the polypeptide or biosimilar thereof is indicated for patients with nasopharyngeal carcinoma. The invention further relates to a safe therapeutic dose of a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof, in combination with docetaxel, for use for treating patients with nasopharyngeal carcinoma. In one embodiment, the polypeptide is aflibercept.

BACKGROUND OF THE INVENTION

A standard therapy for patients with nasopharyngeal carcinoma (NPC) is concurrent cisplatin chemotherapy, optionally followed by adjuvant chemotherapy with cisplatin and fluorouracil. Intensity-modulated radiation therapy (IMRT) allows relatively good locoregional control. However, the development of distant metastasis (occurring in 30% of patients) remains problematic. Therefore, more effective systemic therapy is still needed to improve overall survival of the patients.

Vascular endothelial growth factor (VEGF) has become a major target for anticancer therapy due to its important role in tumor angiogenesis. The efficacy of targeting VEGF has been proven in several clinical trials with the humanized anti-VEGF monoclonal antibody bevacizumab. The convincing evidence of antitumor activity of anti-VEGF therapy demonstrated that optimization of such an approach might deliver additional improvements in efficacy. In NPC, VEGF overexpression is particularly frequent at about 67%, which is correlated with lower survival.

Aflibercept, a novel anti-angiogenic protein consisting of human VEGF receptor extracellular domains fused to the Fc region of human immunoglobulin 1 , interferes with the biologic actions of VEGF by combining with VEGF and preventing it from interacting with its receptors on endothelial cells. Aflibercept has been shown to bind to VEGF-A much more potently than does anti-VEGF monoclonal antibody; moreover, aflibercept binds to the related pro-angiogenic factors VEGF-B and placental growth factor (PIGF). In preclinical studies, aflibercept has shown antitumor effects and anti-angiogenic activity as a single agent and in combination with cytotoxic agents in patients with breast, colorectal and gynecologic cancers. Aflibercept is anticipated to be more active than other anti- VEGF agents because of its particularly high affinity for binding to VEGF and its ability to bind to other related proangiogenic factors such as VEGF-B and PIGF.

The emergence of docetaxel and docetaxel-based chemotherapy regimens has substantially improved the clinical outcome for patients with head and neck cancer as well as NPC. However, new and improved treatment options are still desperately needed. Clinical trial data support the efficacy of aflibercept in combination with docetaxel in patients with ovarian, primary peritoneal and fallopian tube cancers (Coleman et al. 201 1 Lancet Oncol. 12, 1 109-1 1 17). A phase I dose-escalation study of aflibercept in combination with docetaxel, TCD6120, has been conducted recently in western patients with breast, colorectal, gynecologic and lung tumors (Isambert et al. 2012 Clin. Cancer Res. 18, 1743-1750).

The inventors have now determined that aflibercept is appropriate for treating patients with advanced NPC, and identified a suitable treatment regimen for treating patients with NPC with a combination of aflibercept and docetaxel .

DESCRIPTION OF THE INVENTION

The invention relates to fusion proteins comprising Ig domain 2 of the FMS-like tyrosine kinase 1 (Flt1 ), Ig domain 3 of Fetal liver kinase 1 (Flk1 ) and a multimerizing component, in particular aflibercept, combined preparations, or compositions, for the use as disclosed herein, as well as articles of manufacture, dosages and methods as disclosed herein.

In one aspect, the invention provides a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, for use for treating patients with nasopharyngeal carcinoma.

In a second aspect, the invention concerns a combined preparation comprising a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, and docetaxel, for simultaneous, separate or sequential use for treating patients with nasopharyngeal carcinoma.

The invention also concerns a pharmaceutical composition comprising a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, and docetaxel, for use for treating patients with nasopharyngeal carcinoma.

In a third aspect, the invention relates to an article of manufacture comprising: a) a packaging material,

b) a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, and

c) a label or package insert contained within said packaging material indicating that the fusion protein or biosimilar thereof is indicated for patients with nasopharyngeal carcinoma. A fourth object of the invention is a safe therapeutic dose of a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, in combination with docetaxel, for use for treating patients with nasopharyngeal carcinoma, wherein said safe therapeutic dose is 6 mg/kg or below.

The invention also pertains to a method of treating nasopharyngeal carcinoma in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or biosimilar thereof.

Fusion proteins

As used herein, the terms "protein" and "polypeptide" are equivalent. The fusion proteins as disclosed herein are fusion or chimeric polypeptides comprising (a) the amino acid sequence of Ig domain 2 of the extracellular domain of a first VEGF receptor, Flt1 , and the amino acid sequence of Ig domain 3 of the extracellular domain of a second VEGF receptor, Flk1 , and (b) a multimerizing component. The fusion proteins as disclosed herein are preferably capable of binding a VEGF polypeptide, and/or of antagonizing VEGF activity.

As used herein, the extracellular domain is defined as the portion of a receptor that, in its native conformation in the cell membrane, is oriented extracellularly where it can contact with its cognate ligand. The extracellular ligand binding domain does not include the hydrophobic amino acids associated with the receptor's transmembrane domain or any amino acids associated with the receptor's intracellular domain. Generally, the intracellular or cytoplasmic domain of a receptor is usually composed of positively charged or polar amino acids (i.e. lysine, arginine, histidine, glutamic acid, aspartic acid). The preceding 15-30, predominantly hydrophobic or apolar amino acids (i.e. leucine, valine, isoleucine, and phenylalanine) comprise the transmembrane domain. The extracellular domain comprises the amino acids that precede the hydrophobic transmembrane stretch of amino acids. Usually the transmembrane domain is flanked by positively charged or polar amino acids such as lysine or arginine.

The fusion protein may be a fusion polypeptide wherein amino acid sequence of Ig domain 2 of the extracellular domain of Flt1 is upstream of the amino acid sequence of Ig domain 3 of the extracellular domain of Flk1 , or a fusion polypeptide wherein the amino acid sequence of Ig domain 2 of the extracellular domain of Flt1 is downstream of the amino acid sequence of Ig domain 3 of the extracellular domain of Flk1.

The multimerizing component may comprise an immunoglobulin domain. For instance, the immunoglobulin domain may be selected from the group consisting of the Fc domain of IgG, the heavy chain of IgG, and the light chain of IgG.

In one embodiment, the fusion protein is aflibercept or ziv-aflibercept. As used herein, aflibercept refers to a dimer also referred to as VEGFR1 R2-Fc. DELTA. C1 or Flt1 D2.Flk1 D3.Fc.DELTA.C1 , in which each dimer comprising two identical monomers, each of which is a fusion protein comprising the signal sequence of VEGFR1 fused to the D2 Ig domain of the VEGFR1 receptor, itself fused to the D3 Ig domain of the VEGFR2 receptor, in turn fused to the Fc domain of lgG1. The amino acid sequence of the monomer is provided in the sequence listing as SEQ ID NO: 1. In one embodiment, the fusion protein as disclosed herein is a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof.

The U.S. Food and Drug Administration (FDA) approved aflibercept under the trade name EYLEA® for the treatment of patients with neovascular (wet) age-related macular degeneration (AMD). In particular, EYLEA® is the trade name for aflibercept as generated, processed and formulated for intravitreal injection.

In light of aflibercept's approved use in treating AMD, the FDA requested that a different name (ziv-aflibercept) be given for the compound's use in the treatment of cancer. Thus, ziv-aflibercept is the United States Adopted Name (USAN) accepted by FDA to designate a pharmaceutical composition comprising aflibercept as generated, processed and formulated for injection via intravenous infusion. Ziv-aflibercept has been approved by the FDA for sale under the tradename ZALTRAP® for the treatment of metastatic colorectal cancer. ZALTRAP® and EYLEA® are obtained by slightly different processes. They both contain aflibercept or ziv-aflibercept, but the ratio of aggregates of aflibercept or ziv- aflibercept is slightly different in ZALTRAP® and EYLEA®.

The European Medicines Agency (EMA) did not request separate names for the compound. Thus, in the European Union the name "aflibercept" is used regardless of the indication.

The production process of aflibercept is typical for a recombinant-Fc fusion protein. In an embodiment the upstream process includes expansion of the CHO host cells and expression of recombinant aflibercept. The downstream process involves clarification and purification of the protein from the culture medium.

The manufacturing process may be initiated with the thawing and inoculation of one working cell bank (WCB) vial. The cell culture is expanded until reaching sufficient density for inoculation into the production bioreactor.

The downstream process typically consists of several chromatography steps (protein A affinity chromatography, Cation exchange chromatography, Anion exchange chromatography and Hydrophobic Interaction chromatography), and includes viral inactivation and filtration steps to clear potential adventitious viral agents. After processing through the step of concentration/diafiltration, the drug substance is filled into containers and stored frozen.

The protein as disclosed herein may also be a biosimilar of a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component.

As used herein, the term "biosimilar" refers to a product approved for the treatment of a disease indication (e.g., cancer) under article 10(4) of Directive 2001/83/EC or under 42 U.S.C. 262(k), or under an equivalent statute of another jurisdiction. Preferably, the biosimilar is approved for the treatment of nasopharyngeal carcinoma.

The biosimilar used in the present invention may be any biosimilar of aflibercept or ziv-aflibercept, whatever is the name given in the registers (such as INN or USAN name), as far it has the sequence SEQ ID NO: 1 , or consists of dimer of this sequence or of a sequence having at least 97 % identity with SEQ ID NO: 1.

The protein chain of the fusion protein as disclosed herein may be glycosylated, with N-acetyl-glucosamine, fucose, galactose, mannose and sialic acids contributing to the carbohydrate structures. The N-linked oligosaccharides consist of mainly bi-antennary structures with zero, one or two terminal sialic acids. Thus in one embodiment, the fusion protein as disclosed herein, or the biosimilar thereof, is sialilated. The fusion protein as disclosed herein, or the biosimilar thereof, may for instance contain about 8 to 12 moles of sialic acid/moles of polypeptide.

Combined preparation and compositions

In a second aspect, the invention concerns a combined preparation, or a combination, comprising a fusion protein or a biosimilar thereof, as defined in the section "fusion proteins", and docetaxel, for simultaneous, separate or sequential use. Docetaxel, also referred to by the trade name Taxotere, is a clinically well- established anti-mitotic chemotherapy medication used mainly for the treatment of breast, ovarian, prostate, and non-small cell lung cancer. Docetaxel has an FDA approved claim for treatment of patients who have locally advanced, or metastatic breast or non small-cell lung cancer who have undergone anthracycline-based chemotherapy and failed to stop cancer progression or relapsed, and a European approval for use in hormone-refractory prostate cancer.

The expression "combined preparation" refers to any preparation comprising at least two components, such as e.g. a fusion protein or a biosimilar thereof, as defined in the section "fusion proteins", and docetaxel. The different components of the combined preparation, or of the combination, may be used simultaneously, semi-simultaneously, separately, sequentially or spaced out over a period of time so as to obtain the maximum efficacy of the combination.

As a result, for the purposes of the present invention, the combined preparations or combinations are not exclusively limited to those which are obtained by physical association of the constituents, but also to those which permit a separate administration, which can be simultaneous or spaced out over a period of time.

Alternatively, the different components may be co-formulated. Therefore, the combined preparations or combinations of the present invention also encompass pharmaceutical compositions containing the combinations as disclosed herein, i.e. pharmaceutical compositions comprising a fusion protein or a biosimilar thereof, as defined in the section "fusion proteins", and docetaxel. As appreciated by skilled artisans, the combined preparations or combinations or compositions as disclosed herein are suitably formulated to be compatible with the intended route of administration.

The pharmaceutically acceptable formulation comprising the fusion protein or biosimilar thereof may be a freeze-dried or lyophilized formulation. Lyophilized formulations can be reconstituted into solutions, suspensions, emulsions, or any other suitable form for administration or use. Lyophilized formulations are typically first prepared as liquids, then frozen and lyophilized. The lyophilization process is well known to those of ordinary skill in the art, and typically includes sublimation of water from a frozen formulation under controlled conditions.

Lyophilized formulations are typically reconstituted for use by addition of an aqueous solution to dissolve the lyophilized formulation. A wide variety of aqueous solutions can be used to reconstitute a lyophilized formulation. Preferably, lyophilized formulations are reconstituted using water. Lyophilized formulations may be reconstituted with a solution consisting essentially of water (e.g., USP WFI, or water for injection) or bacteriostatic water (e.g., USP WFI with 0.9% benzyl alcohol). However, solutions comprising buffers and/or excipients and/or one or more pharmaceutically acceptable carriers can also be used.

The liquid formulation used to generate a freeze-dried or lyophilized formulation may for instance comprise the fusion protein or biosimilar thereof in a pharmaceutically effective amount, a buffer, a stabilizer, and a bulking agent. Freeze-dried or lyophilized formulations may comprise histidine, since histidine, in comparison to phosphate, is more effective at stabilizing the fusion protein when the fusion protein is lyophilized. Organic co- solvents, such as PEG 3350, may be used to stabilize the fusion protein when agitated, mixed, or handled. A lyoprotectant may be used in freeze-dried or lyophilized formulations. Lyoprotectants help to maintain the secondary structure of proteins when freeze-dried or lyophilized. Two example lyoprotectants are glycine and sucrose, which may be used together.

Alternatively, the pharmaceutically acceptable formulation comprising the fusion protein or biosimilar thereof may also be a liquid formulation. The liquid formulation may comprise a pharmaceutically effective amount of the fusion protein. The formulation can also comprise one or more pharmaceutically acceptable carriers, buffers, bulking agents, stabilizers, preservatives, and/or excipients. An example of a pharmaceutically acceptable liquid formulation comprises the fusion protein or biosimilar thereof in a pharmaceutically effective amount, a buffer, a co-solvent, and one or more stabilizers.

The liquid formulation may comprise phosphate buffer, an organic co-solvent, and one or more thermal stabilizers to minimize formation of aggregates and low molecular weight products when stored, and about 10 mg/ml to about 50 mg/ml fusion protein, wherein the formulation is from about pH 6.0-6.5. The liquid formulation may for instance comprise sucrose, sodium chloride, sodium citrate dihydrate, citric acid monohydrate, polysorbate 20, sodium phosphate dibasic heptahydrate, sodium phosphate monobasic monohydrate, sodium hydroxide and/or hydrochloric acid and water for injections. The liquid formulation may for instance comprise about 5 mM phosphate buffer, about 5 mM citrate, about 100 mM NaCI, about 25% sucrose, and about 10-50 mg/ml fusion protein, wherein the formulation is at a pH of about 6.0; optionally polysorbate may be present (e.g., 0.1 % polysorbate 20). Although either NaCI or sucrose can be used as a stabilizer, a combination of NaCI and sucrose has been established to stabilize the fusion protein more effectively than either individual stabilizer alone.

In one embodiment, the pharmaceutically acceptable formulation is hyperosmolar. In particular, the osmolarity of the combined preparations or combinations or compositions as disclosed herein may be higher than 500 mOsm.

Article of manufacture In a third aspect, the invention relates to an article of manufacture comprising: a) a packaging material,

b) a fusion protein or a biosimilar thereof as defined in the section "fusion proteins", and

c) a label or package insert contained within said packaging material indicating that the fusion protein or biosimilar thereof is indicated for patients with nasopharyngeal carcinoma.

In one embodiment, the label or package insert further indicates that the fusion protein or biosimilar thereof, in combination with docetaxel, is indicated for patients with nasopharyngeal carcinoma.

Medical indications The fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein may be used for the treatment of nasopharyngeal carcinoma.

Another aspect of the invention concerns a method of treating nasopharyngeal carcinoma in a patient in need thereof, said method comprising administering to said patient a therapeutically effective amount of a fusion protein or a biosimilar thereof as defined in the section "fusion protein". Another aspect of the invention concerns a method of treating nasopharyngeal carcinoma in a patient in need thereof, said method comprising administering to said patient therapeutically effective amounts of a fusion protein or a biosimilar thereof as defined in the section "fusion protein", and docetaxel.

The invention further pertains to the use of a fusion protein or a biosimilar thereof as defined in the section "fusion protein" for the manufacture of a medicament, a combined preparation or a pharmaceutical composition intended to treat nasopharyngeal carcinoma. "Treating" or "treatment" of carcinoma as used herein includes limiting disease severity, preventing development of carcinoma, i.e. for instance preventing increase of a tumour, or preventing spread of cancer, i.e. for instance preventing formation of metastasis, and palliating symptoms of cancer.

The term "carcinoma" designates any disease involving unregulated proliferation of epithelial cells, and which may result in unregulated cell growth, lack of differentiation, tumours formation, local tissue invasion, and/or metastasis formation. In the context of the invention, the carcinoma is nasopharyngeal carcinoma, and preferably locoregionally advanced nasopharyngeal carcinoma. According to one embodiment, the carcinoma to be treated is resistant to or has progressed following prior radiotherapy, chemotherapy and/or surgery. In this context, the fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein may advantageously be used as a second line treatment.

A "prophylactically or therapeutically effective amount" refers to a quantity of a composition that confers a prophylactic or therapeutic effect on the treated subject. The therapeutic effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. subject gives an indication of or feels an effect). Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. In the context of the invention, the fusion protein or biosimilar thereof may be administered at a dosage comprised between 1 mg/kg and 10 mg/kg. In particular, the fusion protein or biosimilar thereof may be administered at a dosage of about 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg. In one embodiment, the fusion protein or biosimilar thereof is administered at a dosage of 4 to 8 mg/kg, in particular 5 to 7 mg/kg, or about 6 mg/kg.

Thus, another aspect of the invention concerns a safe therapeutic dose of the fusion protein or a biosimilar thereof, in combination with docetaxel, for use for treating patients with nasopharyngeal carcinoma, wherein said safe therapeutic dose is comprised between 1 mg/kg and 10 mg/kg. In particular, the safe therapeutic dose is of about 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 6 mg/kg, 7 mg/kg, 8 mg/kg, 9 mg/kg, or 10 mg/kg. In one embodiment, the safe therapeutic dose is of 4 to 8 mg/kg, in particular of 5 to 7 mg/kg, or of about 6 mg/kg or below.

As used herein, the terms "administration" and "co-administration" refer to administration of the fusion protein (or biosimilar thereof) and docetaxel concurrently, i.e. simultaneously in time, or sequentially, i.e. administration of the fusion protein (or biosimilar thereof) followed by administration of docetaxel, or administration of docetaxel followed by administration of the fusion protein (or biosimilar thereof). After administration of the fusion protein or docetaxel, the other component can be administered substantially immediately thereafter or after an effective time period. The effective time period is the amount of time given for realization of maximum benefit from the administration of the components. Alternatively, the fusion protein (or biosimilar thereof) and docetaxel may be co-formulated.

The combined preparations or combinations or compositions as disclosed herein are compositions which can be administered parentally and, in particular, intravenously.

It is possible for each administration to vary in its duration from a rapid administration to a continuous perfusion. In one embodiment, the fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein is administered as an intravenous infusion. The fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein may be administered every week, every 2 weeks or every 3 weeks. In one embodiment, the fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein is administered as an intravenous infusion over 30 minutes to 2 hours, in particular over about 1 hour.

According to another aspect of the invention, the fusion protein or biosimilar thereof, optionally comprised in a combined preparation or a pharmaceutical composition as disclosed herein, is administered in combination with 50-100 mg/m², in particular 75 mg/m², of docetaxel.

In one embodiment, the fusion protein or biosimilar thereof is administered intravenously at a dosage of 1-10 mg/kg, for instance 6 mg/kg, in combination with 50-100 mg/m², in particular 75 mg/m², of docetaxel, over 1 hour every 3 weeks.

Patients

In the context of the invention, a "subject in need thereof" is a human who suffers from, or is susceptible of suffering from, nasopharyngeal carcinoma. In one embodiment, the subject in need thereof has a tumour or suffers from nasopharyngeal carcinoma.

According to one embodiment, the subject has already been treated for nasopharyngeal carcinoma. For instance, said subject may have been previously treated with chemotherapy, radiotherapy and/or surgery. The fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein may advantageously be used when said prior treatment or therapy has failed.

According to another embodiment, the subject has higher distant tumor burden or distant metastases.

Also, the fusion protein or biosimilar thereof, the combined preparation or the pharmaceutical composition as disclosed herein may advantageously be used for subjects who are positive for Epstein-Barr virus (EBV). BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 1 shows the amino acid sequence of one of the two monomers forming aflibercept. BRIEF DESCRIPTION OF THE FIGURES

Figure 1 : Mean free and vascular endothelial growth factor (VEGF)-bound aflibercept concentrations-time profiles at cycle 1 (semi-logarithmic scale). Figure 2: Individual and mean [±standard deviation (SD)] free to vascular endothelial growth factor (VEGF)-bound aflibercept C_troug_h ratio over cycles.

Figure 3: Individual and mean [±standard deviation (SD)] endogenous free vascular endothelial growth factor (VEGF) for aflibercept at predose cycle 1 day 1 and at 90-day follow-up visit. LLOQ, lower limit of quantification. Patient No. 156001024 presented with very high concentrations of docetaxel during the entire time course from 1 .5 hours to 24 hours postdose compared with the other patients.

EXAMPLES Example 1 : Materials and methods

Example 1.1: Eligibility

Patients aged 18 years or older with a histologically or cytologically confirmed solid malignancy that was metastatic or unresectable for which standard treatment had failed or who had not been eligible for standard treatment due to safety reasons, but for whom docetaxel treatment was appropriate, were eligible. Patients had to have adequate hematologic, liver, and renal function. Patients were excluded if they had an Eastern Cooperative Oncology Group (ECOG) performance status >1 ; had failed to recover to Grade <1 from any toxic effects of prior anticancer therapy (except alopecia), according to Cancer Institute Common Toxicity Criteria for Adverse Events (NCI CTCAE), version 3.0; or had received treatment with chemotherapy, targeted therapy, hormonal therapy, radiotherapy, surgery, or an investigational agent within 28 days (42 days for nitrosourea agents, mitomycin C, or immunotherapy) of study enrollment. Patients who had received cumulative radiation therapy to >25% of the total bone marrow or who had a history of brain metastases, spinal cord compression, or new evidence of brain or leptomeningeal disease on computed tomography or magnetic resonance imaging at screening were excluded. Patients with uncontrolled hypertension within 4 weeks of study enrollment, myocardial infarction, severe or unstable angina pectoris, coronary or peripheral coronary artery bypass graft surgery, or congestive heart failure were also excluded, as were pregnant or breast-feeding patients or those with bleeding diathesis or underlying coagulopathy.

All patients signed an informed consent form that was approved by all of the relevant institutional ethics committees, and the study was conducted in accordance with the Declaration of Helsinki and good clinical practices. Example 1.2: Study Design and Treatment

This was an open-label, single-center, dose-escalation study of aflibercept administered in combination with docetaxel in patients with advanced solid malignancies to confirm the dose of aflibercept used in western studies based on dose-limiting toxicity (DLT) during cycle 1. When the selected dose of intravenous (IV) infusion of aflibercept in combination with docetaxel was determined, the safety profile, pharmacokinetics (PK), antitumor activity, and other characteristics of that dose of aflibercept were explored in an expansion cohort to determine a recommended dose of aflibercept for further clinical development. During the dose-escalation phase, aflibercept was administered to patients at a starting dose of 4 mg/kg by IV infusion over 1 hour on day 1. The selection of 4 mg/kg as the starting dose in this study was based on the western phase I study, TCD6120. Dose escalation of aflibercept was permitted in 2 mg/kg increments to a maximum of 6 mg/kg. The dose of docetaxel was fixed at 75 mg/m2 throughout the study. Enrollment at the next dose level of aflibercept (6 mg/kg) was not to proceed before≥3 patients treated at the current dose level had received≥1 cycle of study treatment and were evaluated for DLT. The cohort was expanded to 6 patients if DLT was observed in only 1 of the 3 patients.

The selected dose of aflibercept was confirmed by both the investigator and sponsor based on the highest dose of aflibercept at which <1 of a maximum of 6 patients experienced DLT during cycle 1 . Another 10 patients as an expansion cohort were enrolled in addition to the current cohort to receive the selected dose of aflibercept in combination with docetaxel every 3 weeks. Patients who withdrew during the first cycle of study treatment for any reason other than a DLT were to be replaced. In total, enrollment of <22 eligible patients was planned. All patients received docetaxel (75 mg/m2) over 1 hour by IV infusion plus the aflibercept dose on day 1 in 3-week cycles until objective evidence of disease progression, unacceptable toxicity, or withdrawal of consent. Dexamethasone was given before and after administration of docetaxel. Dose modification of aflibercept and docetaxel was permitted for safety reasons.

Example 1.3: Definition of Dose-Limiting Toxicity

DLT was defined as any of the following events during the first cycle of study treatment, based on the NCI CTCAE, version 3.0: Grade 4 febrile neutropenia; neutropenic infection (ie, infection with Grade 3 or 4 neutropenia); Grade 4 neutropenia >7 days; Grade 4 thrombocytopenia, or Grade 3 thrombocytopenia complicated by hemorrhage. Grade 3 nonhematologic toxicities, except fatigue, anorexia, nausea, vomiting, or diarrhea, unless the investigator and sponsor concluded that such inclusion was necessary; Grade 4 nonhematologic toxicities; uncontrolled hypertension as defined by a systolic blood pressure (BP) >150 mm Hg or diastolic BP >100 mm Hg (or a systolic BP >180 mm Hg or a diastolic BP >90 mm Hg if the patient had a history of isolated systolic hypertension) despite 4 weeks of medical management; urinary protein excretion of >3.5 g/24 h (in the case of a urine protein:creatinine ratio of >1 , a 24-hour urine collection was to be obtained) that did not recover to <2.0 g/24 h within 2 weeks; and symptomatic arterial thromboembolic events, including new onset or worsening of preexisting angina, transient ischemic attacks, myocardial infarctions, and cerebrovascular accidents.

Example 1.4: Pharmacokinetic Study

Free aflibercept and VEGF-bound aflibercept assays were conducted under the responsibility of Regeneron Pharmaceuticals, Inc. (Tarrytown, NY), in the Preclinical Department/Sample Analysis Group. Free and VEGF-bound plasma levels of aflibercept were measured on days 1 , 2, 8, and 15 of cycle 1 after aflibercept and on day 1 of cycle 2 using a validated direct enzyme-linked immunosorbent-based assay. Docetaxel concentrations were measured in plasma on day 1 of every cycle by a validated electrospray liquid chromatography mass spectrometry method with a lower limit of quantification of 1 .00 ng/mL.

Example 2: Results A total of 25 patients were screened and 20 patients were eligible for the study, including 4 patients in the 4 mg/kg group and 16 in the 6 mg/kg group. One patient in the 4 mg/kg group was excluded for DLT assessment because of receiving other anticancer therapies during cycle 1. Therefore, the DLT evaluable population included 19 patients. All 20 patients were included in the treated population for other safety- and efficacy-related evaluations. The study population consisted of 3 females and 17 males, with a median age of 45.5 years (range: 22-63) and an ECOG performance status of 0 or 1 . Most of the patients enrolled had NPC (n = 16), and 4 patients had lung cancer . All patients had received prior chemotherapy, with a median of 3 prior anticancer regimens in the 4 mg/kg aflibercept group and 2 regimens in the 6 mg/kg aflibercept group. No patient had prior surgery. Patient and tumor characteristics at baseline are show in Table 1 .

Table 1. Patient and Tumor Characteristics at Baseline

Abbreviations: ECOG, Eastern Cooperative Oncology Group; PS, performance status. Example 2.1: Safety Evaluation

In the dose-escalation phase, 3 patients were planned for enrollment into the first dose level of 4 mg/kg. One patient in the 4 mg/kg group was excluded for DLT assessment because of receiving other anticancer therapies during cycle 1 . Therefore, another patient was recruited into the first dose level. No DLT was reported in cycle 1 in the first 3 patients evaluable for DLT at the 4 mg/kg dose according to the DLT definition at the dose-escalation stage. The next dose level was 6 mg/kg; 1 DLT (neutropenic infection) was observed at that dose level. Consequently, 3 additional patients were enrolled, with no DLT observed. In view of these results, the selected dose of aflibercept in combination with docetaxel was 6 mg/kg. An additional 10 patients were included as an expansion cohort together with the existing 6 patients to receive the selected dose of aflibercept. Among these 10 patients in the expansion cohort, 4 reported AEs during cycle 1 that also fulfilled the definition of a DLT.

All 20 patients experienced≥1 treatment-emergent AE (TEAE) (all grades) possibly related to treatment, of whom 15 (1 patient in the 4 mg/kg group and 14 patients in the 6 mg/kg group) reported Grade≥3 TEAEs possibly related to treatment. One patient in the 4 mg/kg group and 7 patients in the 6 mg/kg group experienced 1 serious TEAE. One AE reported by a patient in the 6 mg/kg group possibly led to death. Fourteen patients (2 of 4 in the 4 mg/kg group and 12 of 16 in the 6 mg/kg group) had≥1 AE leading to permanent treatment discontinuation. The most common TEAEs in both dosing groups (all grades) were stomatitis and dysphonia. Stomatitis was the most frequently reported all-grade TEAE at the 4 mg/kg dose level (3 patients, 75%), and the most second most frequently reported TEAE at the 6 mg/kg dose level (1 1 patients, 68.8%). At the 6 mg/kg dose level, oropharyngeal pain was the most frequently reported all-grade TEAE (12 patients, 75.0%) and the most frequently reported Grade ≥3 TEAE (6 patients, 37.5%). A summary of TEAEs with an incidence of≥10% in≥1 dose group is presented in Table 2.

Table 2. Treatment-Emergent Adverse Events for Aflibercept Occurring With an Incidence of≥10% in≥1 Dose Group

Adverse Event, n (%) Aflibercept

4 mg/kg (n = 4) 6 mg/kg (n = 16)

Grade 3/4 All Grades Grade 3/4 All Grades

Any adverse event, n (%) 1 (25) 4 (100) 14 (87.5) 16 (100)

Oropharyngeal pain 0 1 (25.0) 6 (37.5) 12 (75.0)

Stomatitis 1 (25.0) 3 (75.0) 5 (31.3) 1 1 (68.8)

Alopecia 0 1 (25.0) 0 9 (56.3)

Dysphonia 0 2 (50.0) 0 7 (43.8)

Insomnia 0 1 (25.0) 0 6 (37.5)

Neutropenic infection 0 0 5 (31.3) 5 (31 .3)

Hypertension 0 2 (50.0) 0 4 (25.0)

Cough 0 2 (50.0) 0 4 (25.0)

Epistaxis 0 0 0 4 (25.0)

Decreased weight 0 2 (50.0) 0 3 (18.8)

Headache 0 1 (25.0) 0 3 (18.8)

Diarrhea 0 1 (25.0) 0 2 (12.5)

Arthralgia 0 1 (25.0) 0 2 (12.5)

Noncardiac chest pain 0 1 (25.0) 0 2 (12.5)

Constipation 0 0 0 2 (12.5)

Decreased appetite 0 0 0 2 (12.5)

Hemoptysis 0 0 0 2 (12.5)

Neutropenia 0 0 2 (12.5) 2 (12.5)

Febrile neutropenia 1 (25.0) 1 (25.0) 1 (6.3) 1 (6.3)

Tinnitus 0 1 (25.0) 0 1 (6.3)

Vision blurred 0 1 (25.0) 0 0

Hypoacusis 1 (25.0) 1 (25.0) 0 0 Example 2.2: Study Treatment Exposure

A total of 14 cycles of treatment were administered to the 4 mg/kg group and 45 cycles to the 6 mg/kg group. The median number of cycles by patient for both treatment groups was 3.5 at each dose level. A total of 10 patients received≥4 cycles. No patient required a dose reduction. A summary of exposure to aflibercept and docetaxel by dose level is presented in Table 3. The median relative dose intensity (RDI) of aflibercept was almost the same in each dose group: 0.98 for 4 mg/kg and 1.0 for 6 mg/kg. A similar finding for docetaxel was observed between groups, with an RDI of 0.98 for the 4 mg/kg dose and 1.00 for the 6 mg/kg dose (Table 3). Table 3. Median Exposure, Actual Dose Intensity, and Relative Dose Intensity by

Aflibercept Dose Level

Aflibercept

4 mg/kg 6 mg/kg (n = 4) (n = 16)

Total number of cycles 14 45

Aflibercept, median (range)

Number of infusions 3.5 (1-6) 3.5 (1-4)

Dose intensity, mg/kg/week 1 .31 (1 .1-1 .3) 2.00 (1 .7-2.0)

Relative dose intensity 0.98 (0.8-1.0) 1 .00 (0.8-1 .0)

Docetaxel, median (range)

Number of infusions 3.5 (1-6) 3.5 (1-4)

Relative dose intensity 0.98 (0.8-1.0) 1 .00 (0.8-1 .0)

Example 2.3: Antitumor Activity

Sixteen (12 patients with NPC and 4 with lung cancer) of 20 patients were evaluable for best overall response, including 4 patients in the 4 mg/kg group and 12 patients in the 6 mg/kg group. Two patients (both with NPC) had a partial response and 1 patient (with lung cancer) had stable disease at the 4 mg/kg dose, whereas 9 patients (7 with NPC and 2 with lung cancer) had a partial response and 2 patients (both with NPC) had stable disease at the 6 mg/kg dose (Table 4). Overall, in 12 evaluable patients with NPC, the response rate was 75% (9/12) and the rate of disease control was 92% (1 1/12). Of 8 patients with NPC, 5 (62.5%) who had lung metastasis experienced tumor cavitation during the course of treatment. No tumor cavitation was observed at baseline.

Table 4. Antitumor Activity of Aflibercept

Example 2.4: Pharmacokinetic Evaluation

Overall, PK evaluation was performed in a total of 20 patients, 4 patients at 4 mg/kg and 16 patients at 6 mg/kg. Superimposed curves of mean free and VEGF-bound aflibercept concentration versus time profiles observed after the first administration (cycle 1 ) are illustrated by dose level in Figure 1. Between 4 mg/kg and 6 mg/kg, the concentration of free aflibercept increased with increasing dose; the concentration of VEGF-bound aflibercept appeared to increase in a similar manner. The trough plasma concentration of mean free and VEGF-bound aflibercept had not reached steady state at the end of cycle 3. However, the individual trough concentration (Chough) of free-to-VEGF- bound ratios was relatively constant over treatment cycles. At cycle 4, individual values remained at <1 in the 4 mg/kg dose group and were >1 in 3 of 5 patients in the 6 mg/kg dose group (Figure 2), suggesting that the available circulating VEGF was blocked by aflibercept at doses >4 mg/kg administered every 3 weeks. PK parameters for free and VEGF-bound aflibercept for cycle 1 are summarized in Table 5. From 4 mg/kg to 6 mg/kg (a 1.5-fold increase in dose), testing for mean exposure of free aflibercept found an 1 .8- fold increase in C_max levels and a 1.7-fold increase in area under the concentration- time curve (AUC). In contrast, AUCi_ast of VEGF-bound aflibercept remained constant. Clearance of free aflibercept decreased slightly from 4 mg/kg to 6 mg/kg (1 .44-1.17 L/day). Terminal half-life was approximately 5 days. Different doses of aflibercept did not affect the PK profile of docetaxel. Individual and mean endogenous free VEGF concentrations are summarized in Figure 3. The mean (standard deviation) endogenous free VEGF concentration at baseline was 33.26 pg/mL (33.61 ), and at the follow-up visit 90 days after the last treatment, it was approximately 2-fold higher, at 58.75 pg/mL (48.90).

Table 5. Geometric mean [CV%] Free and VEGF-Bound Aflibercept Pharmacokinetic Parameters for Cycle 1

a Median (min-max).

bOne patient was excluded from the descriptive statistics. Abbreviations: AUC, area under the concentration versus time curve; AUC_0-2i_, area under the concentration versus time curve from day 0 to day 21 ; AUCi_ast, area under the concentration versus time curve from time 0 to ; C_max, maximum observed plasma concentration; CL, clearance; CV, coefficient of variation; t_1/2z, terminal elimination half-life; tiast, time corresponding to the last plasma concentration above the limit of quantification; t_max time of maximum plasma concentration; VEGF, vascular endothelial growth factor; V_ss, volume of distribution at steady state.

Example 2.5: Immunogenicity

Of 20 patients treated with aflibercept, 18 were evaluable for immunogenicity analysis. The results of all serum samples collected from these patients were negative for anti-aflibercept antibodies.

Example 3: Discussion

The results of this study demonstrate that the selected dose of 4 mg/kg to 6 mg/kg of aflibercept in combination with docetaxel is appropriate for patients with advanced solid tumors. Aflibercept plus docetaxel was generally well tolerated in patients. The toxicity profile of patients in this study is similar to that reported in the TCD6120 study and consistent with docetaxel treatment and with aflibercept when used alone. AEs related to VEGF blockade include epistaxis, hemoptysis, and hypertension, which are similar to those seen with other anti-VEGF agents. However, in this study, these AEs were mild with a low incidence. The most common nonhematologic AEs in both dosing groups were stomatitis and dysphonia. The most frequently reported all-grade AEs at 6 mg/kg were oropharyngeal pain, stomatitis, alopecia, dysphonia, insomnia, and neutropenic infection. The most frequently reported grade ≥3/4 AEs at 6 mg/kg were oropharyngeal pain, stomatitis, neutropenic infection, and neutropenia. Hemorrhage had been reported as a well-known complication of antiangiogenesis agents, especially in patients with squamous cancer. In several phase II trials of VEGF receptor tyrosine kinase inhibitors, fatal hemorrhage was reported in patients with NPC. However, in the current study, only two patients experienced grade I and II hemoptysis, and the overall incidence (12.5%) was similar to that found in the phase I studies of aflibercept. During this study, antitumor activity of aflibercept was observed, with 1 1 patients

(55%) experiencing partial response (PR) and 3 (15%) having stable disease (SD). Among the 4 patients who received aflibercept at the 4 mg/kg dose level, 2 patients had a PR and 1 had SD. At the 6 mg/kg dose level, the response rate was 56.3%, with PR recorded in 9 patients and SD in 2 patients. Given the high level of VEGF expression in NPC, the enrollment was mainly focused on NPC. This study enrolled 16 patients with NPC. To the inventors' knowledge, this is first reported phase I study of aflibercept that has evaluated this type of tumor. Overall, in evaluable patients in the NPC group, the response rate was 75% and rate of disease control was 92%. Interestingly, the inventors found that 62.5% of patients with lung metastasis experienced tumor cavitation during the course of treatment, which provides evidence of the anti-angiogenesis activity of aflibercept in patients with NPC. Although this study enrolled heavily pretreated patients, the encouraging antitumor activity of this combination therapy is still comparable with historically reported first-line platinum-based chemotherapy in patients with NPC.

The PK analysis found that the free-to-VEGF-bound aflibercept ratio reached >1 at the 6 mg/kg dose, suggesting that free aflibercept was sufficient to bind with VEGF in the dosing period. In addition, the PK of aflibercept plus docetaxel were similar to the PK observed with each agent when given alone, and the PK of docetaxel were not affected by aflibercept. Based on the DLTs and the overall safety profile, administration of 6 mg/kg aflibercept in combination with 75 mg/m2 docetaxel every 3 weeks was considered appropriate in patients.

In summary, aflibercept plus docetaxel has antitumor activity and is well tolerated in patients with advanced solid malignancies.

Claims

1 . A fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, for use for treating patients with nasopharyngeal carcinoma.

2. A combined preparation comprising a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, and docetaxel, for simultaneous, separate or sequential use for treating patients with nasopharyngeal carcinoma.

3. A pharmaceutical composition comprising a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, and docetaxel, for use for treating patients with nasopharyngeal carcinoma.

4. The fusion protein or biosimilar of claim 1 , the combined preparation of 2, or the pharmaceutical composition of claim 3, wherein the fusion protein or biosimilar thereof is administered at a dosage comprised between 1 mg/kg and 10 mg/kg.

5. The fusion protein or biosimilar of claim 1 or 4, the combined preparation of 2 or 4, or the pharmaceutical composition of claim 3 or 4, wherein the fusion protein or biosimilar thereof is administered at a dosage of 6 mg/kg.

6. The fusion protein or biosimilar of any one of claims 1 or 4-5, the combined preparation of any one of claims 2 or 4-5, or the pharmaceutical composition of any one of claims 3-5, wherein the fusion protein or biosimilar thereof is administered as an intravenous infusion.

7. The fusion protein or biosimilar of any one of claims 1 or 4-6, the combined preparation of any one of claims 2 or 4-6, or the pharmaceutical composition of any one of claims 3-6, wherein the fusion protein or biosimilar thereof is administered every 3 weeks.

8. The fusion protein or biosimilar of any one of claims 1 or 4-7, the combined preparation of any one of claims 2 or 4-7, or the pharmaceutical composition of any one of claims 3-7, wherein the fusion protein or biosimilar thereof is administered at a dosage of 6 mg/kg as an intravenous infusion over 1 hour every 3 weeks.

9. The fusion protein or biosimilar of any one of claims 1 or 4-8, the combined preparation of any one of claims 2 or 4-8, or the pharmaceutical composition of any one of claims 3-8, wherein the fusion protein or biosimilar thereof is administered in combination with 75 mg/m² of docetaxel.

10. An article of manufacture comprising:

a) a packaging material,

1 1. The article of manufacture of claim 10 wherein the label or package insert further indicates that the fusion protein or biosimilar thereof, in combination with docetaxel, is indicated for patients with nasopharyngeal carcinoma.

12. The fusion protein or biosimilar of any one of claims 1 or 4-9, the combined preparation of any one of claims 2 or 4-9, the pharmaceutical composition of any one of claims 3-9, or the article of manufacture of claim 10 or 1 1 , wherein said nasopharyngeal carcinoma is locoregionally advanced nasopharyngeal carcinoma.

13. The fusion protein or biosimilar of any one of claims 1 , 4-9 or 12, the combined preparation of any one of claims 2, 4-9 or 12, the pharmaceutical composition of any one of claims 3-9 or 12, or the article of manufacture of any one of claims 10-

12, wherein said nasopharyngeal carcinoma is resistant to or has progressed following prior radiotherapy, chemotherapy and/or surgery.

14. The fusion protein or biosimilar of any one of claims 1 , 4-9 or 12-13, the combined preparation of any one of claims 2, 4-9 or 12-13, the pharmaceutical composition of any one of claims 3-9 or 12-13, or the article of manufacture of any one of claims 10-13, wherein said fusion protein is a polypeptide comprising SEQ ID NO: 1 or a biosimilar thereof.

15. The fusion protein or biosimilar of any one of claims 1 , 4-9 or 12-13, the combined preparation of any one of claims 2, 4-9 or 12-13, the pharmaceutical composition of any one of claims 3-9 or 12-13, or the article of manufacture of any one of claims 10-13, wherein said fusion protein is aflibercept or ziv-aflibercept, or a biosimilar thereof.

16. A safe therapeutic dose of a fusion protein comprising Ig domain 2 of Flt1 , Ig domain 3 of Flk1 and a multimerizing component, or a biosimilar thereof, in combination with docetaxel, for use for treating patients with nasopharyngeal carcinoma, wherein said safe therapeutic dose is 6 mg/kg or below.