Pharmaceutical compositions and methods of use thereof

Abstract

Provided herein are pharmaceutical compositions comprising an active agent, the active agent being a soluble VEGFR-3 capture molecule. Also provided herein are methods of treatment and uses relating to the pharmaceutical compositions, in particular to methods of treatment and uses relating to ocular diseases and disorders, as well as port devices comprising the pharmaceutical compositions.

Description

[0001] This patent application claims priority to U.S. Provisional Patent Application No. 63 / 374,366, filed on September 1, 2022, the entire contents of which are incorporated herein by reference. Technical Field

[0002] The present disclosure relates to pharmaceutical compositions comprising an active agent, which is a soluble VEGFR-3 capture molecule. The present disclosure also relates to methods and uses of the pharmaceutical compositions, particularly methods and uses of eye diseases and disorders, and port devices comprising the pharmaceutical compositions. Background Art

[0003] The loss or deterioration of vision is an extremely debilitating condition that can have a profound impact on an individual's quality of life. Age-related macular degeneration (AMD) is the leading cause of severe visual impairment in the elderly. The "wet" form of the condition develops when abnormal blood vessels grow into the macula, leaking blood or fluid, leading to macular scarring and vision loss. Another serious eye condition associated with leaky blood vessels is diabetic macular edema (DME), in which leaking fluid due to blood vessel damage accumulates in the macula in individuals with diabetic retinopathy.

[0004] Current treatments for AMD and DME include treatment with laser therapy (e.g., laser photocoagulation), and administration of drugs including ranibizumab. Aflibercept Brolucizumab and corticosteroids (such as triamcinolone).

[0005] The vascular endothelial growth factor (VEGF) protein and its receptor play important roles in angiogenesis (the development of the embryonic vasculature from early differentiated endothelial cells), vasculogenesis (the process of forming new blood vessels from pre-existing vessels), and lymphangiogenesis (the process of forming new lymphatic vessels). Dysfunction of the endothelial cell regulatory system is also a key feature of cancer and many other diseases associated with abnormal angiogenesis, angiogenesis, and lymphangiogenesis.

[0006] Therapies directed at blocking VEGF / PDGF signaling through its receptors have been approved for the treatment of ocular disorders (including AMD and DME) and cancer. For example, the above-mentioned aflibercept is an inhibitor of VEGF, which consists of portions of the extracellular domains of human VEGF receptors 1 and 2 fused to the Fc portion of human IgG1. It acts by binding to circulating VEGF-A and VEGF-B and placental growth factor (PlGF), which normally binds to VEGFR-1 and VEGFR-2 and is therefore a VEGFR-1 / VEGFR-2 capture molecule.

[0007] Another treatment being developed for the treatment of eye disorders is OPT-302, which is a VEGFR-3 capture molecule comprising a portion of the extracellular domain of human VEGF receptor 3 that is soluble in fluids such as blood and plasma and binds to circulating VEGF proteins that normally bind to VEGFR-3, namely VEGF-C and VEGF-D. OPT-302 has completed a Phase 2b clinical trial for wet age-related macular degeneration (wet AMD) and a Phase 2a clinical trial for DME, and a Phase 3 clinical trial for wet AMD is ongoing. Soluble VEGFR-3 capture molecules such as OPT-302 are described, for example, in WO2014 / 124487 and WO2015 / 123715, the entire contents of which are incorporated herein by reference.

[0008] However, drug discovery and development is a long and complex process, and after the therapeutic agent is determined, there are usually significant obstacles to bringing the drug to market and obtaining approval to treat patients. For example, there may be significant challenges in developing pharmaceutical formulations of active agents that provide desired properties, such as safety, with acceptable activity stability, acceptable formulation stability, maintaining sufficient activity over time, facilitating administration, and avoiding application site reactions. For some therapeutic agents, despite efforts to determine suitable formulations, special storage conditions may be required to achieve acceptable formulation life, such as low or ultra-low temperature conditions.

[0009] There remains a need for further pharmaceutical products to treat conditions such as wet AMD and DME.There also remains a need for formulations of soluble VEGFR-3 capture molecules (such as OPT-302) that provide good properties, such as those related to storage stability.

[0010] It should be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms part of the common general knowledge in the art, in Australia or any other country. Summary of the invention

[0011] OPT-302, a soluble VEGFR-3 capture molecule, has a tendency to form dimers when present in some aqueous formulations, associated with loss of binding activity and purity. For example, it was found that the developed aqueous compositions of OPT-302 rapidly formed high levels of OPT-302 dimers and required storage at very low temperatures (e.g., -20°C) to achieve an acceptable shelf life.

[0012] Research has now identified a drug formulation for OPT-302 that provides unexpectedly good stability properties and does not require storage at -20°C to achieve acceptable product longevity.

[0013] Thus, in a first aspect, there is provided an aqueous pharmaceutical composition comprising:

[0014] an active agent which is a soluble VEGFR-3 capture molecule, the active agent being present at a concentration of 5 mg / mL to 250 mg / mL;

[0015] Trehalose;

[0016] Buffering agents; and

[0017] water;

[0018] wherein the pH of the aqueous pharmaceutical composition is 6.5 to 8.0;

[0019] And wherein the pharmaceutical composition comprises trehalose at a concentration of at least 7.0% w / v and / or the pharmaceutical composition does not comprise added sodium chloride.

[0020] In some embodiments, the pharmaceutical composition does not comprise added sodium chloride.

[0021] In some embodiments, the pharmaceutical composition comprises trehalose at a concentration of at least 7.0% w / v.

[0022] In some embodiments, trehalose is present in a concentration of up to 20% w / v. In some embodiments, trehalose is present in a concentration of 8.5% w / v to 15% w / v. In some embodiments, the composition comprises about 10.9% w / v trehalose.

[0023] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, wherein the ligand binding polypeptide comprises immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally has one or more modifications in the N-glycan region of the extracellular domain.

[0024] In some embodiments, the ligand-binding polypeptide comprises an amino acid sequence defined by positions 25 to 329 of SEQ ID NO: 1, provided that the positions of the polypeptide corresponding to positions 104 to 106 of SEQ ID NO: 1 are not the same as NXS or NXT; wherein the ligand-binding polypeptide retains four N-glycosylation sequence segment sites corresponding to positions 33 to 35 of SEQ ID NO: 1, positions 166 to 168 of SEQ ID NO: 1, positions 251 to 253 of SEQ ID NO: 1, and positions 299 to 301 of SEQ ID NO: 1, and is glycosylated at the four N-glycosylation sequence segment sites.

[0025] In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99 to 330 of SEQ ID NO:2.

[0026] In some embodiments, the soluble VEGFR-3 capture molecule has the amino acid sequence shown in any one of SEQ ID NOs:3 to 6, or has the amino acid sequence defined by positions 1 to 536 of SEQ ID NO:3, or has the amino acid sequence defined by positions 1 to 536 of SEQ ID NO:4, or has the amino acid sequence defined by positions 1 to 546 of SEQ ID NO:5, or has the amino acid sequence defined by positions 1 to 546 of SEQ ID NO:6.

[0027] In some embodiments, the ligand-binding polypeptide comprises an amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1; wherein the ligand-binding polypeptide retains five N-glycosylation sequence segment sites corresponding to positions 33 to 35 of SEQ ID NO:1, positions 104 to 106 of SEQ ID NO:1, positions 166 to 168 of SEQ ID NO:1, positions 251 to 253 of SEQ ID NO:1, and positions 299 to 301 of SEQ ID NO:1, and is glycosylated at the five N-glycosylation sequence segment sites.

[0028] In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99 to 330 of SEQ ID NO:2.

[0029] In some embodiments, the soluble VEGFR-3 capture molecule has the amino acid sequence shown in SEQ ID NO:7, or has the amino acid sequence defined by positions 1 to 547 of SEQ ID NO:7.

[0030] In some embodiments, the active agent is present at a concentration of up to 120 mg / mL. In some embodiments, the active agent is present at a concentration of about 40 mg / mL, or about 80 mg / mL, or about 120 mg / mL.

[0031] In some embodiments, the pH of the composition is 7.2 to 7.8. In some embodiments, the pH of the composition is about 7.5.

[0032] In some embodiments, the buffer is sodium phosphate. In some embodiments, the buffer is present at a concentration of 5 mM to 100 mM. In some embodiments, the buffer is present at a concentration of up to 50 mM. In some embodiments, the buffer is present at a concentration of about 10 mM.

[0033] In some embodiments, the composition comprises a surfactant. In some embodiments, the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate. In some embodiments, the surfactant is present at a concentration of 0.005% to 0.2% w / v. In some embodiments, the surfactant is present at a concentration of about 0.01% w / v.

[0034] In some embodiments, the osmolarity of the composition is 300mOsm / kg to 1000mOsm / kg. In some embodiments, the osmolarity of the composition is 350mOsm / kg. In some embodiments, the osmolarity of the composition is 400mOsm / kg. In some embodiments, the osmolarity of the composition is 400mOsm / kg to 600mOsm / kg.

[0035] In some embodiments, the composition is substantially free of sodium chloride.

[0036] In some embodiments, the composition comprises no additional sugars.

[0037] In some embodiments, the composition does not include an additional tonicity adjusting agent.

[0038] In some embodiments, the composition consists essentially of:

[0039] an active agent at a concentration of about 40 mg / ml, which is a soluble VEGFR-3 capture molecule comprising a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain;

[0040] Trehalose at a concentration of about 10.9% w / v;

[0041] Sodium phosphate at a concentration of about 10 mM;

[0042] Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and

[0043] water;

[0044] The pH of the aqueous pharmaceutical composition is about 7.5.

[0045] In another aspect, there is provided a lyophilized pharmaceutical composition for reconstitution comprising:

[0046] an active agent, which is a soluble VEGFR-3 capture molecule;

[0047] Trehalose; and

[0048] Buffers;

[0049] The weight ratio of trehalose to the active agent is 1:3 to 40:1.

[0050] In some embodiments, the weight ratio of trehalose to active agent is 1: 1 to 7.5: 1, 1: 1 to 5: 1, or 2.1: 1 to 4.5: 1. In some embodiments, the weight ratio of trehalose to active agent is about 2.7:1.

[0051] In some embodiments, the buffer is sodium phosphate. In some embodiments, the buffer is sodium phosphate, and the weight ratio of sodium phosphate to active agent is 1:3 to 1:1000, or 1:3 to 1:200, or 1:5 to 1:100. In some embodiments, the buffer is sodium phosphate, and the weight ratio of sodium phosphate to active agent is about 0.03:1.

[0052] In some embodiments, the composition comprises a surfactant. In some embodiments, the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

[0053] In another aspect, there is also provided a reconstituted pharmaceutical composition, wherein the pharmaceutical composition is obtained by mixing the lyophilized pharmaceutical composition as defined herein with an aqueous diluent.

[0054] In some embodiments of the above defined aspects, the pharmaceutical composition is formulated for intravitreal injection.

[0055] In another aspect, there is provided a method of inhibiting neovascularization in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein.

[0056] In another aspect, there is provided a method of treating and / or preventing a disease or disorder associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein.

[0057] In another aspect, there is provided the use of a VEGF-C and / or VEGF-D capture molecule or a salt thereof for the preparation of a pharmaceutical composition as defined herein for the treatment and / or prevention of a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis.

[0058] In another aspect, there is provided a pharmaceutical composition as defined herein for use in the treatment and / or prevention of a disease or disorder associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis.

[0059] In some embodiments of the methods, uses, and pharmaceutical compositions used, the disease or condition is an ocular disease or condition. In some embodiments of the methods, uses, and pharmaceutical compositions used, the ocular disease or condition is selected from macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion, and macular telangiectasia. In some embodiments of the methods, uses, and pharmaceutical compositions used, the ocular disease or condition is wet age-related macular degeneration. In some embodiments of the methods, uses, and pharmaceutical compositions used, the ocular disease or condition is diabetic macular edema. In some embodiments of the methods, uses, and pharmaceutical compositions used, the pharmaceutical composition is administered in combination with an additional active agent. In some embodiments of the methods, uses, and pharmaceutical compositions used, the additional active agent is an anti-VEGF-A agent or an anti-VEGF-B agent. In some embodiments of the methods, uses, and pharmaceutical compositions used, the additional active agent is selected from ranibizumab, aflibercept, bevacizumab, and brolucizumab.

[0060] In some embodiments of the methods, uses, and pharmaceutical compositions for use, the pharmaceutical composition is administered intravitreally.

[0061] In some embodiments of the methods, uses, and pharmaceutical compositions for use, the pharmaceutical composition is administered using a port device implanted in the eye, the port device comprising a reservoir for the pharmaceutical composition and allowing controlled release of the active agent into the vitreous of the eye.

[0062] In another aspect, there is also provided a port device for implantation in an eye, the port device comprising a reservoir containing a pharmaceutical composition as defined herein, and wherein the port device allows controlled release of the active agent into the vitreous of the eye.

[0063] In some embodiments, the port device includes a semipermeable membrane that allows passive diffusion of the active agent into the vitreous body of the eye.

[0064] In some embodiments, the port device includes a septum that allows the reservoir to be refilled with additional drug composition using a needle. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Graphs showing the results of a stability study of OPT-302 compositions according to the present disclosure and comparative compositions are shown. The levels of % high molecular weight active agent species formed by the compositions over time were determined at 37°C.

[0066] Figure 2 Shown are graphs showing the results of a stability study of OPT-302 compositions according to the present disclosure. The level of % active agent monomer formation was determined for the compositions over time at 25°C.

[0067] Figure 3 Shown are graphs showing the results of a stability study of OPT-302 compositions according to the present disclosure. The level of % active agent monomer formation was determined for the compositions over time at 5°C. DETAILED DESCRIPTION

[0068] definition

[0069] Throughout this specification, unless the context requires otherwise, the word "comprises / comprising" or variations thereof will be understood to imply the inclusion of stated elements, integers or steps, or groups of elements, integers or steps, but not the exclusion of any other elements, integers or steps, or groups of elements, integers or steps.

[0070] As used herein, the term "and / or", such as "X and / or Y", should be understood to mean either or both of "X and Y" and "X or Y", and should be considered to provide explicit support for both or either meanings.

[0071] Unless indicated to the contrary, the term "about" as used herein means + / - 10%, more preferably + / - 5%, of the specified value.

[0072] As used herein, unmodified nouns include both the singular and plural aspects unless the context clearly indicates otherwise.

[0073] As used herein, the phrase "at least one of" when used with a list of items means that different combinations of one or more of the listed items may be used, and only one of the items in the list may be required.

[0074] As used herein, the term "subject" refers to any organism susceptible to a disease or condition. For example, a subject can be an animal, a mammal, a primate, a livestock animal (e.g., sheep, cattle, horses, pigs), a companion animal (e.g., dogs, cats), or a laboratory animal (e.g., mice, rabbits, rats, guinea pigs, hamsters). In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal.

[0075] As used herein, the term "treating" and variations thereof include one or more of curing the disease or condition, reducing the severity of the disease or condition, preventing or slowing the progression of the disease or condition, and alleviating the symptoms associated with the disease or condition.

[0076] As used herein, the term "preventing" and variations thereof include one or more of preventing a subject from developing a disease or disorder, delaying the onset of a disease or disorder, and preventing the disorder or condition.

[0077] As used herein, the term "therapeutically effective amount" means that the pharmaceutical composition comprising a soluble VEGFR-3 capture molecule is administered in an amount sufficient to treat or prevent the disorder or condition being treated.

[0078] Term " identity " as known in the art refers to the relationship between the sequence of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing sequences.In the art, " identity " also means the sequence relatedness degree of nucleic acid molecules or polypeptide sequences, which is determined as the case may be, as determined by the matching between the strings of two or more nucleotide sequences or two or more amino acid sequences.By the specific mathematical model (i.e. " algorithm ") of computer program, gap comparison (if any), " identity " measures the percentage of identical matching between the smaller of two or more sequences.Suitable algorithms for determining the percentage identity of the present disclosure include BLASTP and BLASTN, using the most common and recognized default parameters.

[0079] The term "component domain" as used herein refers to a domain within a ligand binding molecule that is derived from or based on a protein domain within the extracellular portion of a receptor protein. For example, each Ig domain of VEGFR-3 (D1-D7) constitutes a component domain. The component domains mentioned herein include both complete natural wild-type domains and insertions, deletions and / or substitution variants that substantially retain the functional characteristics of the complete domain. It will be apparent to those skilled in the art that many variants of the above-mentioned domains (e.g., Ig-domains) can be obtained that retain substantially the same functional characteristics as the wild-type domain.

[0080] Active Agent

[0081] The pharmaceutical compositions of the present disclosure comprise an active agent which is a soluble VEGFR-3 capture molecule.

[0082] Vascular endothelial growth factor receptor 3 (VEGFR-3; formerly known as Flt4) is a receptor for VEGF-C and VEGF-D ligands and is primarily found in vascular and lymphatic endothelial cells. It is primarily involved in angiogenesis and lymphangiogenesis. Soluble VEGFR-3 capture molecules can be used to treat diseases and conditions associated with neovascularization and / or vascular permeability, abnormal angiogenesis and / or lymphangiogenesis, such as many ocular conditions, including wet age-related macular degeneration and diabetic macular edema. It can also be applied to other disease indications associated with abnormal angiogenesis and / or lymphangiogenesis (e.g., cancer).

[0083] As used herein, the term "soluble" in relation to an active agent means that the active agent has a sufficiently high solubility in biological fluids such as blood, plasma and / or vitreous humor so that it can be used to bind to circulating VEGF-C. In some embodiments, the solubility of the soluble VEGFR-3 capture molecule in plasma is at least 1 mg / mL, or at least 2 mg / mL, or at least 5 mg / mL, or at least 10 mg / mL, or at least 20 mg / mL, or at least 30 mg / mL, or at least 40 mg / mL.

[0084] Active agent is VEGFR-3 capture molecule.As indicated herein, VEGFR-3 capture molecule is a molecule capable of binding to circulating VEGF-C and / or VEGF-D.In some embodiments, soluble VEGFR-3 capture molecule binds to human VEGF-C with a KD of about 1 nM or less (e.g., 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less).In some embodiments, soluble VEGFR-3 capture molecule binds to human VEGF-D with a KD of about 5 nM or less (e.g., 2 nM, 1 nM, 500 pM, 400 pM, 300 pM, 200 pM, 100 pM, 50 pM, 10 pM or less).

[0085] The binding affinity for VEGF-C and VEGF-D can be determined by any suitable assay. For example, the binding affinity can be determined using ELISA or using surface plasmon resonance. Such techniques are described, for example, in WO 2014 / 124487A1, the entire contents of which are incorporated herein by reference.

[0086] In some embodiments, the soluble VEGFR-3 capture molecule is or comprises a polypeptide.

[0087] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide. For example, the ligand binding polypeptide can be a fragment of a growth receptor tyrosine kinase extracellular domain (ECD) or can comprise a fragment of a growth receptor tyrosine kinase extracellular domain (ECD). In some embodiments, the fragment can differ from the wild-type sequence in a manner that does not eliminate growth factor binding, and the fragment is preferably modified in a manner described herein to improve its properties as a therapeutic agent for administration to an object / patient in need thereof.

[0088] VEGF-C and VEGF-D bind with high affinity to at least one VEGF receptor (or receptor heterodimer) selected from VEGFR-2 and VEGFR-3 and stimulate its phosphorylation. Preferred ligand binding polypeptides do not simply bind to their target growth factors. Preferred ligand binding polypeptides also inhibit the phosphorylation of at least one (preferably all) receptor tyrosine kinases to which the growth factor to which it binds stimulates the growth factor to bind. The stimulation of tyrosine phosphorylation is easily measured using in vitro cell-based assays and antibodies.

[0089] A ligand binding polypeptide that is "specific" for a particular growth factor is a ligand binding molecule that specifically recognizes the active form of the growth factor (e.g., the form that circulates in the body). Preferably, the ligand binding polypeptide also specifically binds to other forms of the growth factor. For example, VEGF-C (and VEGF-D) is translated as a prepro-molecule with a large number of amino-terminal and carboxyl-terminal propeptides, which are cleaved to produce a "fully processed" form of VEGF-C (or VEGF-D) that binds to and stimulates VEGFR-2 and VEGFR-3. A ligand binding polypeptide that is specific for VEGF-C (or VEGF-D) binds to at least the fully processed form of VEGF-C (or VEGF-D), and preferably also to partially processed and unprocessed forms.

[0090] SEQ ID NO: 1 comprises the amino acid sequence of human VEGFR-3, wherein positions 1 to 24 of SEQ ID NO: 1 correspond to the putative signal peptide, and positions 25 and onwards of SEQ ID NO: 1 correspond to the putative mature form of the receptor lacking the putative signal peptide.

[0091] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide comprising a portion of the extracellular domain (ECD) of human VEGFR-3. The ECD of human VEGFR-3 comprises 7 immunoglobulin-like domains. Domains 1 to 3 are involved in ligand binding, and domains 4 to 7 are involved in the structural rearrangement necessary for receptor dimerization.

[0092] The complete ECD of VEGFR is not required for ligand (growth factor) binding. The ECD of VEGFR-3 has six complete Ig-like domains, and one cleaved Ig-like domain - D5 of VEGFR-3 is post-translationally cleaved into disulfide-linked subunits, leaving VEGFR-3 (Veikkola, T., et al., Cancer Res. 60: 203-212 (2000)). In some embodiments, a receptor fragment comprising at least the first three Ig-like domains of the family is sufficient to bind to the ligand. Soluble receptors capable of binding to VEGF-C and VEGF-D, thereby inhibiting VEGF-C or VEGF-D activity or signal transduction via VEGFR-3 are also disclosed in WO2000 / 023565, WO2000 / 021560, WO2002 / 060950 and WO2005 / 087808, the disclosures of which are incorporated herein by reference in their entirety. Those soluble receptors, optionally with modifications described herein, are contemplated as soluble VEGFR-3 capture molecules of the present disclosure.

[0093] The following table defines the approximate boundaries of the Ig-like domain of human VEGFR-3. These boundaries are important because the boundaries selected can be used to form ligand binding molecules and can therefore affect the binding properties of the resulting constructs.

[0094]

[0095] In some embodiments, the ligand binding polypeptide comprises a portion of the amino acid sequence of at least one of immunoglobulin-like domains 1, 2, and 3 of the ECD of human VEGFR-3.

[0096] In some embodiments, the ligand binding polypeptide comprises substantially the entire amino acid sequence or the entire amino acid sequence of at least one of immunoglobulin-like domains 1, 2, and 3 of the ECD of human VEGFR-3.

[0097] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide, which is a purified or isolated ligand binding polypeptide comprising a first amino acid sequence, which has at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity to the amino acid sequence defined by positions 47 to 115 of SEQ ID NO: 1 or positions 25 to 115 of SEQ ID NO: 1. The foregoing fragments of SEQ ID NO: 1 correspond approximately to or include the first immunoglobulin-like domain of the extracellular domain (ECD) of human VEGFR-3 ("D1 of VEGFR-3").

[0098] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide, which is a purified or isolated ligand binding polypeptide comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 47 to 115 of SEQ ID NO: 1 or positions 25 to 115 of SEQ ID NO: 1, provided that the positions of the polypeptide corresponding to positions 104 to 106 of SEQ ID NO: 1 are not the same as NXS or NXT (X represents any amino acid).

[0099] The Ig-like domains 1 to 3 of VEGFR-3 contain five putative N-glycosylation sites (referred to herein as N1, N2, N3, N4, and N5 sequence segments of VEGFR-3, respectively). N1 corresponds to amino acid positions 33 to 35 of SEQ ID NO: 1; N2 corresponds to amino acid positions 104 to 106 of SEQ ID NO: 1; N3 corresponds to amino acid positions 166 to 168 of SEQ ID NO: 1; N4 corresponds to amino acid positions 251 to 253 of SEQ ID NO: 1, and N5 corresponds to amino acid positions 299 to 301 of SEQ ID NO: 1. In some embodiments, the ligand binding molecules described herein comprise modifications in the N2 sequence segment of the molecule.

[0100] In some embodiments, the putative glycosylation stretch at positions 104 to 106 is eliminated from the amino acid sequence of the ligand binding polypeptide. The term "eliminated" as used herein means that the primary amino acid sequence is changed (by substitution, deletion or insertion) at at least one position to destroy the NXT stretch motif. In one variant, the amino acid corresponding to position 104 of SEQ ID NO: 1 may be deleted and replaced with another amino acid (e.g., glutamine, aspartic acid, glutamic acid, arginine and lysine).

[0101] For example, in some embodiments, the amino acid in the ligand binding molecule corresponding to position 104 of SEQ ID NO: 1 is deleted and replaced with another amino acid. Conservative substitutions are preferred. In some embodiments, the amino acid corresponding to position 104 of SEQ ID NO: 1 is deleted and replaced with an amino acid selected from glutamine, aspartic acid, glutamic acid, arginine and lysine. In some embodiments in which the N2 sequence segment of SEQ ID NO: 1 is modified as described above, the N1, N3, N4 and N5 sequence segments of SEQ ID NO: 1 are preferably unchanged in amino acid sequence.

[0102] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide, which is a purified or isolated ligand binding polypeptide sequence comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 154 to 210 of SEQ ID NO: 1. The amino acid sequence defined by the positions corresponding to the polypeptide at positions 154 to 210 corresponds approximately to or comprises the second immunoglobulin-like domain of the ECD of human VEGFR-3 ("D2 of VEGFR-3").

[0103] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide, which is a purified or isolated ligand binding polypeptide sequence comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 248 to 314 of SEQ ID NO: 1. The amino acid sequence defined by the positions corresponding to the polypeptide at positions 248 to 314 corresponds approximately to or includes the third immunoglobulin-like domain of the ECD of human VEGFR-3 ("D3 of VEGFR-3").

[0104] In some embodiments, the ligand binding polypeptide comprises substantially all or all of the amino acid sequence of immunoglobulin-like domains 1 and 2 of the ECD of human VEGFR-3. In some embodiments, the ligand binding polypeptide comprises substantially all or all of the amino acid sequence of immunoglobulin-like domains 2 and 3 of the ECD of human VEGFR-3. In some embodiments, the ligand binding polypeptide comprises substantially all or all of the amino acid sequence of immunoglobulin-like domains 1, 2, and 3 of the ECD of human VEGFR-3.

[0105] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain.

[0106] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide, which is a purified or isolated ligand binding polypeptide sequence comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1, or is identical to the amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1.

[0107] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising an amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1; wherein the ligand-binding polypeptide retains five N-glycosylation sequence segment sites corresponding to positions 33 to 35 of SEQ ID NO:1, positions 104 to 106 of SEQ ID NO:1, positions 166 to 168 of SEQ ID NO:1, positions 251 to 253 of SEQ ID NO:1, and positions 299 to 301 of SEQ ID NO:1, and is glycosylated at the five N-glycosylation sequence segment sites.

[0108] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide, which is a purified or isolated ligand binding polypeptide sequence comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 25 to 329 of SEQ ID NO: 1, or is identical to the amino acid sequence defined by positions 25 to 329 of SEQ ID NO: 1, provided that the positions of the polypeptide corresponding to positions 104 to 106 of SEQ ID NO: 1 are not the same as NXS or NXT (X represents any amino acid).

[0109] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand-binding polypeptide comprising an amino acid sequence defined by positions 25 to 329 of SEQ ID NO:1, provided that the position of the polypeptide corresponding to positions 104 to 106 of SEQ ID NO:1 is not the same as NXS or NXT; wherein the ligand-binding polypeptide retains four N-glycosylation sequence segment sites corresponding to positions 33 to 35 of SEQ ID NO:1, positions 166 to 168 of SEQ ID NO:1, positions 251 to 253 of SEQ ID NO:1, and positions 299 to 301 of SEQ ID NO:1, and is glycosylated at the four N-glycosylation sequence segment sites.

[0110] In some embodiments, the putative glycosylation stretch at positions 104 to 106 is eliminated from the amino acid sequence of the ligand binding polypeptide. The term "eliminated" as used herein means that the primary amino acid sequence is changed (by substitution, deletion or insertion) at at least one position to destroy the NXT stretch motif. In one variant, the amino acid corresponding to position 104 of SEQ ID NO: 1 may be deleted and replaced with another amino acid (e.g., glutamine, aspartic acid, glutamic acid, arginine and lysine).

[0111] Constructs comprising additional Ig-like domains of VEGFR-3 linked in a manner that produces a ligand binding polypeptide are specifically contemplated. For example, a soluble VEGFR-3 capture molecule may comprise some or substantially all or all of the seven Ig-like domains of VEGFR-3.

[0112] In some embodiments where the ligand binding polypeptide comprises an amino acid sequence that roughly corresponds to two or more component domains of VEGFR-3, the component domains may be directly linked to each other or may be linked via one or more spacers. Preferably, the component domains are linked via one or more spacers.

[0113] For example, the ligand-binding polypeptide may optionally comprise a sequence preceding the N-terminal-most Ig-like domain, between Ig-like domains, and / or following the C-terminal-most Ig-like domain.

[0114] In one embodiment, the spacer comprises one or more peptide sequences of 1 to 100 amino acids, preferably 1 to 50 amino acids, in length between the component domains. In one embodiment, the spacer between the two component domains consists essentially of a peptide sequence naturally linked to the component domains in native VEGFR-3.

[0115] In some embodiments in which the ligand-binding polypeptide comprises an amino acid sequence that roughly corresponds to or comprises consecutive component domains of VEGFR-3 (e.g., D1-D2 or D1-D2-D3), the component domains may be connected via one or more spacers comprising one or more peptide sequences of 1 to 100 amino acids, preferably 1 to 50 amino acids in length, between the component domains.

[0116] In some embodiments, the spacer between two component domains consists essentially of a peptide sequence corresponding to those connecting the corresponding continuous component domains in native VEGFR-3. In some embodiments, the spacer between two continuous component domains comprises an amino acid sequence having at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identity with the amino acid sequence connecting the continuous domains in native VEGFR-3, or it is identical to the amino acid sequence connecting the continuous domains in native VEGFR-3.

[0117] In some embodiments where the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide containing multiple component domains (e.g., component domains D1, D2, and D3 of VEGFR-3), the component domains may be directly connected to each other or may be connected via one or more spacers. Generally, the term "spacer" means one or more molecules, such as nucleic acids or amino acids, or non-peptide moieties, such as polyethylene glycol or disulfide bridges, which may be inserted between one or more component domains to form a covalent bond. Spacer sequences may be used to provide desired destination sites between components for ease of operation. Spacers may also be provided to enhance the expression of ligand binding polypeptides from host cells to reduce steric hindrance so that a component or a group of components may present its / their optimal tertiary structure and / or interact appropriately with its / their target molecule. For spacers and methods for identifying desired spacers, see, e.g., George et al. (2003) Protein Engineering 15: 871-879, which is specifically incorporated herein by reference. The spacer sequence may comprise one or more amino acids naturally attached to the receptor component, or may be an added sequence that is used to enhance the expression of the ligand binding polypeptide, provide a particularly desired destination site, allow the component domain to form an optimal tertiary structure, and / or enhance the interaction of the component or group of components with its / their target molecule. In one embodiment, the spacer comprises one or more peptide sequences between one or more components, and its length is 1 to 100 amino acids, preferably 1 to 50 amino acids. In a preferred embodiment, the spacer between two component domains is essentially composed of amino acids naturally attached to the receptor component in the wild-type receptor. In the case where the ligand binding polypeptide comprises multiple component domains from the same receptor, the domains are adjacent to each other in the natural receptor (e.g., D1, D2, and D3 of VEGFR-3), and in one embodiment, the domains are connected to each other (e.g., D1 to D2 and D2 to D3) using a spacer corresponding to a naturally occurring amino acid linking sequence.

[0118] In some variations, each ligand-binding polypeptide is expressed as a fusion with a fusion partner protein (eg, an immunoglobulin constant region), and the heterologous fusion partners are linked to form a ligand-binding molecule.

[0119] In some embodiments, the ligand binding molecule is a polypeptide comprising a portion of a human VEGFR-3 ECD, wherein the portion binds to one or both of human VEGF-C and human VEGF-D, and comprises at least the first, second, and third Ig-like domains of the VEGFR-3 ECD, wherein the amino acid sequence of the ECD fragment of VEGFR-3 is modified from wild-type VEGFR-3 to eliminate the second putative N-linked glycosylation sequence segment of wild-type VEGFR-3, and wherein the polypeptide lacks VEGFR-3 Ig-like domains 4 to 7, and preferably any transmembrane domain and preferably any intracellular domain.

[0120] In addition to monomer constructs, the disclosure also includes multimeric ligand binding constructs, which include two or more ligand binding molecules described herein that are covalently or non-covalently linked to each other to form a dimer or multimer structure. In some variants, the connection occurs between the VEGFR-3-like sequences of the ligand binding polypeptide; in other variants, the connection occurs between heterologous polypeptides linked to one or both of the VEGFR-3-like sequences.

[0121] References herein to the ligand binding polypeptides described herein include references to variants thereof. In some embodiments, the ligand binding polypeptide is a variant. In other embodiments, the ligand binding polypeptide is not a variant.

[0122] Ligand binding polypeptide can be derived from VEGFR-3, and VEGFR-3 includes splice variants and naturally occurring allelic variants.Allelic variants are well known in the art, and represent a nucleic acid sequence or alternative form comprising the replacement, deletion or addition of one or more nucleotides but not causing any substantial functional changes in the encoded polypeptide. Exemplary allelic variants of VEGFR-3 have been reported in the literature, for example, in http: / / www.uniprot.org / uniprot / P35916, and include the 149th, 378th, 494th, 527th and 641st in the ECD. Standard methods can be easily used to produce such polypeptides, including site-directed mutagenesis of polynucleotides, or specific enzymatic cleavage and connection. Similarly, it is contemplated to use a peptide mimetic compound or a compound in which one or more amino acid residues are replaced by non-natural amino acids or amino acid analogs that retain binding activity.

[0123] Preferably, when using amino acid replacement, the replacement is conservative, i.e., amino acid is replaced by an amino acid with similar size and similar charge characteristics. The term "conservative replacement" used herein means that an amino acid residue is replaced by another biologically similar residue. Some examples of conservative replacement include a hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine replacing another, or a polar residue replacing another, such as arginine replacing lysine, glutamic acid replacing aspartic acid, or glutamine replacing asparagine etc. Neutral hydrophilic amino acids that can replace each other include asparagine, glutamine, serine and threonine. The term "conservative replacement" also includes using the replaced amino acid to replace the unsubstituted amino acid.

[0124] Alternatively, conservative amino acids can be grouped as described in Lehninger, (Biochemistry, 2nd ed.; Worth Publishers, Inc. NY: NY, pp. 71-77 (1975)), as shown below:

[0125] Non-polar (hydrophobic)

[0126] A. Aliphatic: A, L, I, V, P,

[0127] B. Aromatic: F, W,

[0128] C. Sulfur content: M,

[0129] D. Borderline: G.

[0130] Uncharged polarity

[0131] A. Hydroxyl: S, T, Y,

[0132] B. Amide: N, Q,

[0133] C. Thiol: C,

[0134] D. Critical: G.

[0135] Positively charged (alkaline): K, R, H.

[0136] Negatively charged (acidic): D, E.

[0137] Soluble VEGFR-3 capture molecules can, for example, comprise a fusion partner (e.g., such as a heterologous peptide), for example to impart desired properties (e.g., such as increased serum half-life, increased solubility in aqueous media and / or the ability to target specific cell populations, such as tumor cells or retinal cells).

[0138] In some embodiments, the fusion partner is any heterologous component that enhances the functionality of the ligand binding polypeptide. Thus, for example, the fusion partner can improve solubility, regulate clearance, promote targeting of specific cells or tissue types, enhance biological activity, help produce and / or recycle, enhance pharmacological properties, or enhance the pharmacokinetic (PK) profile of the ligand binding polypeptide. Regarding enhancing the PK profile, this can be achieved by, for example, enhancing the serum half-life, tissue permeability, lack of immunogenicity, or stability of the ligand binding molecule. In some embodiments, the fusion partner is selected from a multimerization component, a serum protein, or a molecule capable of binding to a serum protein.

[0139] In some embodiments, the fusion components comprise domains from an immunoglobulin source, such as human IgG, Ig, or IgA.

[0140] In some variants, the soluble VEGFR-3 capture molecule comprises an immunoglobulin constant domain or a fragment thereof. In some embodiments, the soluble VEGFR-3 capture molecule comprises a human immunoglobulin G domain or a fragment thereof. The amino acid sequence of the human immunoglobulin 1 heavy chain constant domain is listed in SEQ ID NO: 2. In some embodiments, the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99 to 330 of SEQ ID NO: 2.

[0141] In some embodiments, the soluble VEGFR-3 capture molecule comprises an immunoglobulin constant domain fragment comprising an amino acid sequence that is at least 80%, or at least 85%, or at least 90%, or at least 92%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% identical to the amino acid sequence defined by positions 99 to 330 of SEQ ID NO:2, or is identical to the amino acid sequence defined by positions 99 to 330 of SEQ ID NO:2.

[0142] In some embodiments, the immunoglobulin constant domain fragment may have an omitted C-terminal amino acid residue.For example, in some embodiments, the immunoglobulin constant domain fragment has an amino acid sequence defined by positions 99 to 329 of SEQ ID NO:2.

[0143] The amino acid sequence derived from immunoglobulin can be connected to the C-terminus or N-terminus of the ligand binding polypeptide, preferably connected to the C-terminus. Cells transfected with DNA encoding immunoglobulin light chain fusion protein and immunoglobulin heavy chain fusion protein express heavy chain / light chain heterodimers comprising respective ligand binding polypeptides. Both ligand binding polypeptides advantageously include natural or heterologous signal peptides when initially synthesized to promote secretion from cells, but the signal sequence can be cut, for example, during secretion. Variants of any of the aforementioned embodiments including signal peptides are contemplated. The natural signal peptide of human VEGFR-3 includes residues 1 to 24 of SEQ ID NO:1. Many other signal peptide proteins are taught in the literature.

[0144] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide fused (as a single polypeptide chain) to the Fc portion of human immunoglobulin G (IgG).

[0145] In some embodiments, the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide fused (as a single polypeptide chain) to the Fc portion of human immunoglobulin G (IgG), wherein there is a single amino acid substitution in the second Ig-like domain to remove the N-glycosylation site.

[0146] In some embodiments, the ligand binding polypeptides described herein optionally comprise a linker that connects a fusion partner (e.g., such as a heterologous peptide) to the ligand binding polypeptide, such as the Factor Xa linker sequence DPIEGRGGGGG (SEQ ID NO: 8). In other embodiments, the ligand binding molecule comprises a polypeptide in which the C-terminal amino acid of the ligand binding polypeptide is directly linked to the N-terminal amino acid of the heterologous peptide fusion partner via a peptide bond. In some embodiments, the ligand binding polypeptide and the heterologous peptide are linked via an amide bond (directly or via a linker polypeptide) to form a single polypeptide chain.

[0147] In some embodiments, the soluble VEGFR-3 capture molecule is OPT-302. OPT-302 has the amino acid sequence shown in SEQ ID NO:3.

[0148] In some embodiments, the soluble VEGFR-3 capture molecule comprises the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 3. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 3.

[0149] In some embodiments, the soluble VEGFR-3 capture molecule comprises the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 4. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 4.

[0150] In some embodiments, the soluble VEGFR-3 capture molecule comprises the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 5. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence defined by positions 1 to 546 of SEQ ID NO: 5.

[0151] In some embodiments, the soluble VEGFR-3 capture molecule comprises the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO: 6. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence defined by positions 1 to 546 of SEQ ID NO: 6.

[0152] In some embodiments, the soluble VEGFR-3 capture molecule is VGX-300. VGX-300 has the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the soluble VEGFR-3 capture molecule comprises the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence consisting of the amino acid sequence shown in SEQ ID NO:7. In some embodiments, the soluble VEGFR-3 capture molecule has an amino acid sequence defined by positions 1 to 546 of SEQ ID NO:7.

[0153] If desired, the soluble VEGFR-3 capture molecules described herein may include a functional region that promotes purification or production. Some specific examples of such additional amino acid sequences include a GST sequence or a His tag sequence. In some variants, in order to formulate a composition for pharmaceutical use, the region that promotes purification is removed.

[0154] The soluble VEGFR-3 capture molecule can be chemically modified (eg, glycosylated, pegylated, etc.) to impart desired properties. Such modifications preferably do not substantially reduce the growth factor binding affinity or specificity of the ligand binding molecule.

[0155] The polypeptides may be modified, for example, by glycosylation, amidation, carboxylation or phosphorylation, or by producing acid addition salts, amides, esters, in particular C-terminal esters and N-acyl derivatives.

[0156] In some embodiments, the soluble VEGFR-3 capture molecules described herein optionally include at least one PEG (polyethylene glycol) portion connected to the molecule. For example, in some embodiments, about 20 to 40kDa PEG is connected to the amino terminus of the ligand binding molecule. Polyethylene glycol used herein means any form of PEG that can be used to derive other proteins, such as mono-(C1-C10) alkoxy- or aryloxy-polyethylene glycol. PEG is a neutral polyether of straight or branched chain, with a wide range of molecular weights to choose from, and is soluble in water and most organic solvents. When present in water, PEG is effective in excluding other polymers or peptides, mainly through its high dynamic chain mobility and hydrophilic properties, thereby generating a water shell or hydration ball when connected to other proteins or polymer surfaces. PEG is non-toxic, non-immunogenic, and is approved for internal consumption by the Food and Drug Administration (FDA).

[0157] The polypeptide can be conjugated to a reporter group, including but not limited to a radiolabel, a fluorescent label, an enzyme (e.g., an enzyme that catalyzes a calorimetric or fluorometric reaction), a substrate, a solid matrix or a carrier (e.g., biotin or avidin). Some examples of analogs are described in WO 98 / 28621 and Olofsson, et al., Proc. Nat'l. Acad. Sci. USA, 95: 1709-1 714 (1998), U.S. Pat. Nos. 5,512,545 and 5,474,982; U.S. Patent Application Nos. 20020164687 and 20020164710, the entire contents of each of which are incorporated herein by reference.

[0158] In some variations, the ligand binding molecule comprises a signal peptide that directs secretion of the molecule from cells expressing the molecule.

[0159] Soluble VEGFR-3 capture molecules can be prepared by any suitable method. For example, a cell line (e.g., a eukaryotic cell line, a Chinese hamster ovary cell line) can be transfected with a vector containing a polynucleotide sequence encoding an amino acid sequence of a soluble VEGFR-3 capture molecule, and the cell line is cultured to express the capture molecule. Methods for producing and purifying soluble VEGFR-3 capture molecules are described in WO2014 / 124487A1, WO2015 / 123715A1, and WO2002 / 060950A1, each of which is incorporated herein by reference in its entirety.

[0160] The soluble VEGFR-3 capture molecule is present in the aqueous pharmaceutical composition at a concentration of 5 mg / mL to 250 mg / mL.

[0161] In some embodiments, the soluble VEGFR-3 capture molecule is present at a concentration starting from 10 mg / mL, 20 mg / mL, 30 mg / mL, 40 mg / mL, 50 mg / mL, 60 mg / mL, 70 mg / mL, 80 mg / mL, 90 mg / mL, 100 mg / mL, 110 mg / mL, 120 mg / mL, 130 mg / mL, 140 mg / mL, 150 mg / mL, 160 mg / mL, 170 mg / mL, 180 mg / mL, 190 mg / mL, 200 mg / mL, 210 mg / mL, 220 mg / mL, 230 mg / mL or 240 mg / mL. In some embodiments, the soluble VEGFR-3 capture molecule is present at a concentration of up to 240 mg / mL, up to 230 mg / mL, up to 220 mg / mL, up to 210 mg / mL, up to 200 mg / mL, up to 190 mg / mL, up to 180 mg / mL, up to 170 mg / mL, up to 160 mg / mL, up to 150 mg / mL, up to 140 mg / mL, up to 130 mg / mL, up to 120 mg / mL, up to 110 mg / mL, up to 100 mg / mL, up to 90 mg / mL, up to 80 mg / mL, up to 70 mg / mL, up to 60 mg / mL, up to 50 mg / mL, up to 40 mg / mL, up to 30 mg / mL, up to 20 mg / mL, or up to 10 mg / mL. Preferably, the soluble VEGFR-3 capture molecule is present in a concentration of up to 120 mg / mL, more preferably up to 100 mg / mL, still more preferably up to 80 mg / mL, still more preferably up to 60 mg / mL, further more preferably up to 40 mg / mL.

[0162] In some embodiments, the soluble VEGFR3 capture molecule is present at a concentration of 5 mg / mL up to 120 mg / mL, 5 mg / mL up to 100 mg / mL, 5 mg / mL up to 80 mg / mL, 5 mg / mL up to 60 mg / mL, 5 mg / mL up to 50 mg / mL, 5 mg / mL up to 40 mg / mL, 10 mg / mL up to 120 mg / mL, 10 mg / mL up to 100 mg / mL, 10 mg / mL up to 80 mg / mL, 10 mg / mL up to 60 mg / mL, 10 mg / mL up to 50 mg / mL, 10 mg / mL up to 40 mg / mL, mL up to 40 mg / mL, 20 mg / mL up to 120 mg / mL, 20 mg / mL up to 100 mg / mL, 20 mg / mL up to 80 mg / mL, 20 mg / mL up to 60 mg / mL, 20 mg / mL up to 50 mg / mL, 20 mg / mL up to 40 mg / mL, 30 mg / mL up to 120 mg / mL, 30 mg / mL up to 100 mg / mL, 30 mg / mL up to 80 mg / mL, 30 mg / mL up to 60 mg / mL, 30 mg / mL up to 50 mg / mL, or 30 mg / mL up to 40 mg / mL.

[0163] In some embodiments, the soluble VEGFR-3 capture molecule is present at a concentration of about 10 mg / mL, about 15 mg / mL, about 20 mg / mL, about 25 mg / mL, about 30 mg / mL, about 35 mg / mL, about 40 mg / mL, about 45 mg / mL, about 50 mg / mL, about 60 mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 110 mg / mL or about 120 mg / mL.

[0164] excipient

[0165] The aqueous pharmaceutical composition of the present disclosure contains trehalose. Trehalose is a disaccharide formed by two glucose units connected by a 1,1-glycosidic bond. Generally, the glucose units present in trehalose are α-glucose units. Trehalose is also called α,α-trehalose, α-D-pyranoglucopyranosyl-(1→1)-α-D-pyranoglucoside, α-D-pyranoglucopyranosyl-α-D-pyranoglucoside and D-(+)-trehalose. The CAS number of trehalose is 6138-23-4. Trehalose has the following chemical structure:

[0166]

[0167] Trehalose is available in anhydrous and dihydrate forms. The molar mass of trehalose in anhydrous form is 342.3 g / mol, and the molar mass of trehalose in dihydrate form is 378.3 g / mol. Trehalose is available from a variety of suppliers, such as Pfanstiel (www.pfanstiehl.com). Trehalose dihydrate is also available from Sigma Aldrich, Merck, Fisher Scientific, Acros, and Alfa Aesar.

[0168] In some embodiments, the aqueous pharmaceutical composition of the present disclosure comprises trehalose at a concentration of at least 7.0% w / v. Regardless of the form of trehalose used to produce the formulation (e.g., anhydrous trehalose or trehalose dihydrate), the % w / v of trehalose is related to the weight percentage of trehalose, and, for example, does not include any related solvates (e.g., dihydrates). For example, a 10% w / v solution of trehalose can be prepared using 10g of anhydrous trehalose and made up to 100ml with water. Alternatively, a 10% w / v solution of trehalose can be prepared using 11.1g of trehalose dihydrate and made up to 100ml with water.

[0169] In some embodiments, trehalose is present at a concentration of up to 20% w / v, or up to 15% w / v, or up to 14% w / v, or up to 13% w / v, or up to 12% w / v, or up to 11% w / v. In some embodiments, trehalose is present at a concentration of at least 7.5% w / v, at least 8.0% w / v, at least 8.5% w / v, at least 9% w / v, at least 9.5% w / v, at least 10% w / v, or at least 10.5% w / v. In some embodiments, trehalose is present in an amount of 7.0% w / v to 20% w / v, or 7.0% w / v to 15% w / v, or 7.0% w / v to 14% w / v, or 7.0% w / v to 13% w / v, or 7.0% w / v to 12% w / v, or 7.5% w / v to 20% w / v, or 7.5% w / v to 15% w / v, or 7.5% w / v to 14% w / v, or 7.5% w / v to 13% w / v, or 7.5% w / v to 12% w / v. w / v to 12% w / v, or 8.0% w / v to 20% w / v, or 8.0% w / v to 15% w / v, or 8.0% w / v to 14% w / v, or 8.0% w / v to 13% w / v, or 8.0% w / v to 12% w / v, or 8.5% w / v to 20% w / v, or 8.5% w / v to 15% w / v, or 8.5% w / v to 14% w / v, or 8.5% w / v to 13% w / v, or 8.5% w / v to 12% w / v 9% w / v to 12% w / v, or 9% w / v to 20% w / v, or 9% w / v to 15% w / v, or 9% w / v to 14% w / v, or 9% w / v to 13% w / v, or 9% w / v to 12% w / v, or 9.5% w / v to 20% w / v, or 9.5% w / v to 15% w / v, or 9.5% w / v to 14% w / v, or 9.5% w / v to 13% w / v, or 9.5% w / v to 12% w / v, or 10 % w / v to 20% w / v, or 10% w / v to 15% w / v, or 10% w / v to 14% w / v, or 10% w / v to 13% w / v, or 10% w / v to 12% w / v, or 10.5% w / v to 20% w / v, or 10.5% w / v to 15% w / v, or 10.5% w / v to 14% w / v, or 10.5% w / v to 13% w / v, or 10.5% w / v to 12% w / v.In some embodiments, trehalose is present at about 7.0% w / v, about 7.5% w / v, about 8.0% w / v, 8.5% w / v, about 9% w / v, about 9.5% w / v, about 10% w / v, about 10.1% w / v, about 10.2% w / v, about 10.3% w / v, about 10.4% w / v, about 10.5% w / v, about 10.6% w / v, about 10.7% w / v, about 10.8% w / v, about 10.9% w / v, about 11% w / v, about 11.1% w / v, about 11.2% w / v, about 11.3% w / v, about 11.4% w / v, about 11.6% w / v, about 11.7% w / v, about 11.8% w / v, about 11.9% w / v, about 11.1% w / v, about 11.2% w / v, about 11.3% w / v, about 11.4% w / v The invention also provides an aqueous solution of at least one molecule of the present invention, wherein the aqueous solution of the present invention is present in a concentration of about 12% w / v, about 11.3% w / v, about 11.4% w / v, about 11.5% w / v, about 11.6% w / v, about 11.7% w / v, about 11.8% w / v, about 11.9% w / v, about 12% w / v, about 12.5% ​​w / v, about 13% w / v, about 13.5% w / v, about 14% w / v, about 14.5% w / v, about 15% w / v, about 16% w / v, about 17% w / v, about 18% w / v, about 19% w / v, or about 20% w / v.

[0170] Aqueous pharmaceutical compositions contain water. Sterile, high-purity water is usually used, such as water for injection.

[0171] Aqueous pharmaceutical compositions include buffers, for example, for maintaining pH within a desired range. Any suitable buffer can be used. Some examples of buffers include phosphate buffers (e.g., sodium dihydrogen phosphate, disodium phosphate), amino acids such as histidine buffers (e.g., histidine hydrochloride), citrate buffers (e.g., sodium citrate), tris (2-amino-2-(hydroxymethyl) propane-1,3-diol) and acetate buffers (e.g., sodium acetate). In some embodiments, the buffer is tris or a phosphate buffer. In some embodiments, the buffer is a phosphate buffer, for example, it can be a mixture of an acidic and alkaline form of phosphate. In some embodiments, the buffer is sodium phosphate, for example, a mixture of sodium dihydrogen phosphate and disodium phosphate.

[0172] Buffer is included in a suitable concentration, for example, it can be present at a concentration of up to 100mM, up to 90mM, up to 80mM, up to 70mM, up to 60mM, up to 50mM, up to 40mM, up to 30mM, up to 20mM, or up to 10mM. In some embodiments, the buffer is present at a concentration of at least 5mM or at least 10mM. In some embodiments, the buffer is present at a concentration of 5mM to 100mM, 5mM to 80mM, 5mM to 70mM, 5mM to 60mM, 5mM to 50mM, 5mM to 40mM, 5mM to 30mM, or 5mM to 20mM. In some embodiments, the buffer is included at a concentration of about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, or about 100 mM.

[0173] It has been found that aqueous pharmaceutical compositions comprising soluble VEGFR-3 capture molecules having a higher pH have improved stability with respect to dimer formation.

[0174] The pH of the aqueous pharmaceutical composition is 6.5 to 8.0. In some embodiments, the pH of the composition is 6.5 to 7.0, 7.0 to 7.5, 7.5 to 8.0, 7.2 to 7.8, 7.3 to 7.7, or 7.4 to 7.6. In some embodiments, the pH of the composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0175] In some embodiments, the aqueous pharmaceutical composition comprises a surfactant. When a surfactant is included, it can be, for example, an ionic surfactant (e.g., a cationic, anionic, or zwitterionic) or a nonionic (e.g., neutral) surfactant. Some examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (e.g., available under the trade name polysorbate 90, Twain Sorbitan monooleate (e.g., sold under the trade name and Twain ), polyethylene glycols, and poloxamers (eg, copolymers of poly(propylene oxide) and poly(ethylene oxide)) (eg, sold under the trade name Pluronic (Pluronic ) is sold as ). In some embodiments, the aqueous pharmaceutical composition comprises a surfactant which is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

[0176] When included, the surfactant may be present, for example, at a concentration of 0.005% to 0.2% w / v. In some embodiments, the surfactant is present at a concentration of 0.005% to 0.1% w / v, or 0.005% to 0.05% w / v, or 0.005% to 0.02% w / v. In some embodiments, the surfactant is present at a concentration of about 0.005% w / v, or about 0.006% w / v, or about 0.007% w / v, or about 0.008% w / v, or about 0.009% w / v, or about 0.01% w / v, or about 0.011% w / v, or about 0.012% w / v, or about 0.013% w / v, or about 0.014%, or about 0.015% w / v, or about 0.016% w / v, or about 0.017% w / v, or about 0.018% w / v, or about 0.019% w / v, or about 0.02% w / v.

[0177] In some other embodiments, the aqueous pharmaceutical composition does not include a surfactant.

[0178] It has been found that aqueous pharmaceutical compositions comprising soluble VEGFR-3 capture molecules and having a relatively high osmolarity provide good stability properties. Osmolarity relates to the concentration of osmotically active particles in a solution and is usually defined in units of mOsm / kg.

[0179] Thus, in some embodiments, the osmolarity of the aqueous pharmaceutical composition is at least 300 mOsm / kg, or at least 350 mOsm / kg, or at least 400 mOsm / kg. In some embodiments, the osmolarity of the aqueous pharmaceutical composition is up to 1000 mOsm / kg, or up to 900 mOsm / kg, or up to 800 mOsm / kg, or up to 700 mOsm / kg, or up to 600 mOsm / kg, or up to 500 mOsm / kg. In some embodiments, the osmolarity of the aqueous pharmaceutical composition is 300 mOsm / kg to 1000 mOsm / kg, or 350 mOsm / kg to 1000 mOsm / kg, or 400 mOsm / kg to 1000 mOsm / kg, or 300 mOsm / kg to 800 mOsm / kg, or 350 mOsm / kg to 800 mOsm / kg, or 400 mOsm / kg to 800 mOsm / kg, or 300 mOsm / kg to 600 mOsm / kg, or 350 mOsm / kg to 600 mOsm / kg, or 400 mOsm / kg to 600 mOsm / kg.

[0180] Aqueous pharmaceutical compositions can contain relatively few components and still provide good stability properties.

[0181] As defined herein, a tonicity agent is a substance that affects the osmolarity of a pharmaceutical composition. A tonicity agent is typically included to adjust the osmolarity of a composition to a desired value.

[0182] While in some embodiments, the aqueous pharmaceutical composition comprises an additional tonicity agent, in other embodiments, the aqueous pharmaceutical composition does not comprise an additional tonicity agent. The term additional tonicity agent refers to a substance that substantially affects the osmolarity of the pharmaceutical composition, and is a substance in addition to the active agent, trehalose, buffer, water, and surfactant (if present). Some examples of tonicity agents include sugars (e.g., sucrose, dextrose), certain salts (e.g., sodium chloride, potassium chloride), polyols (e.g., mannitol, sorbitol, glycerol).

[0183] In some embodiments, the pharmaceutical composition comprises less than 50 mM sodium chloride, or less than 25 mM sodium chloride, or less than 10 mM sodium chloride, or less than 5 mM sodium chloride.

[0184] In some embodiments, the pharmaceutical composition does not contain added sodium chloride. As used herein, the term "does not contain added sodium chloride" means that no sodium chloride is added during the preparation of the pharmaceutical composition. It should be understood that the pharmaceutical composition may still contain trace amounts of sodium chloride, for example, if the pH of the formulation is adjusted by adding hydrochloric acid and sodium hydroxide, some small amounts of sodium chloride may be formed.

[0185] In some embodiments, the aqueous pharmaceutical composition is substantially free of sodium chloride. In some embodiments, the composition comprises less than 1 mM sodium chloride, or comprises less than 0.5 mM sodium chloride, or comprises less than 0.2 mM sodium chloride, or comprises less than 0.1 mM sodium chloride, or comprises less than 0.05 mM sodium chloride. In some embodiments, the aqueous pharmaceutical composition comprises no detectable sodium chloride.

[0186] In some embodiments, the aqueous pharmaceutical composition is substantially free of additional sugars (i.e., it is substantially free of sugars (e.g., monosaccharides or disaccharides) other than trehalose and any sugars that form part of the active ingredient). In some embodiments, the aqueous pharmaceutical composition comprises less than 1 mM of additional sugars, less than 0.5 mM of additional sugars, less than 0.2 mM of additional sugars, less than 0.1 mM of additional sugars, or less than 0.05 mM of additional sugars. In some embodiments, the aqueous pharmaceutical composition comprises no detectable additional sugars.

[0187] In some embodiments, the aqueous pharmaceutical composition does not comprise any components other than water, soluble VEGFR-3 capture molecules, phosphate buffer, and surfactant (except for impurities that may be present in those components).

[0188] In some embodiments, the aqueous pharmaceutical composition comprises the following: an active agent which is a soluble VEGFR-3 capture molecule, the active agent being present at a concentration of 5 mg / mL to 120 mg / mL,

[0189] Trehalose at a concentration of 8.5% w / v to 20% w / v;

[0190] a buffer at a concentration of 5 mM to 20 mM;

[0191] optionally a surfactant; and

[0192] water;

[0193] The pH of the aqueous pharmaceutical composition is 6.5 to 8.0.

[0194] In some embodiments, the aqueous pharmaceutical composition comprises the following: an active agent which is a soluble VEGFR-3 capture molecule, the active agent being present at a concentration of 20 mg / mL to 80 mg / mL,

[0195] Trehalose at a concentration of 9% w / v to 13% w / v;

[0196] a buffer at a concentration of 5 mM to 20 mM, wherein the buffer is sodium phosphate;

[0197] a surfactant which is polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and

[0198] water;

[0199] The pH of the aqueous pharmaceutical composition is 7.0 to 8.0.

[0200] In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of the active agent, trehalose, a buffer, and a surfactant.

[0201] In some embodiments, the aqueous pharmaceutical composition consists of, or consists essentially of, the active agent, trehalose, sodium phosphate buffer, and polyoxyethylene (20) sorbitan monolaurate.

[0202] In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of:

[0203] an active agent at a concentration of about 40 mg / ml, which is a soluble VEGFR-3 capture molecule comprising a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain;

[0204] Trehalose at a concentration of about 10.9% w / v;

[0205] a buffer at a concentration of about 10 mM, wherein the buffer is sodium phosphate;

[0206] Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and

[0207] water;

[0208] The pH of the aqueous pharmaceutical composition is about 7.5.

[0209] In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of:

[0210] an active agent at a concentration of about 40 mg / ml, which is OPT-302 or VGX-300;

[0211] Trehalose at a concentration of about 10.9% w / v;

[0212] a buffer at a concentration of about 10 mM, wherein the buffer is sodium phosphate;

[0213] Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and

[0214] water;

[0215] The pH of the aqueous pharmaceutical composition is about 7.5.

[0216] In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of:

[0217] An active agent at a concentration of about 40 mg / ml, comprising or consisting of the amino acid sequence of any one of SEQ ID NOs: 3, 4, 5, and 6;

[0218] Trehalose at a concentration of about 10.9% w / v;

[0219] a buffer at a concentration of about 10 mM, wherein the buffer is sodium phosphate;

[0220] Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and

[0221] water;

[0222] The pH of the aqueous pharmaceutical composition is about 7.5.

[0223] In some embodiments, the aqueous pharmaceutical composition consists of or consists essentially of:

[0224] an active agent comprising or consisting of the amino acid sequence of SEQ ID NO: 7 at a concentration of about 40 mg / ml;

[0225] Trehalose at a concentration of about 10.9% w / v;

[0226] a buffer at a concentration of about 10 mM, wherein the buffer is sodium phosphate;

[0227] Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and

[0228] water;

[0229] The pH of the aqueous pharmaceutical composition is about 7.5.

[0230] The aqueous pharmaceutical composition may be prepared in advance and provided as a pre-prepared aqueous pharmaceutical formulation to a hospital, operating room, etc. Alternatively, it may be provided as a solid composition (eg, a lyophilized pharmaceutical composition) for reconstitution with water prior to use.

[0231] Therefore, there is also provided a lyophilized pharmaceutical composition for reconstitution comprising:

[0232] an active agent, which is a soluble VEGFR-3 capture molecule;

[0233] Trehalose; and

[0234] Buffers;

[0235] The weight ratio of trehalose to the active agent is 1:3 to 40:1.

[0236] Also provided is a reconstituted pharmaceutical composition, wherein the pharmaceutical composition is obtained by mixing a lyophilized pharmaceutical composition as defined herein with an aqueous diluent.

[0237] In use, an appropriate amount of aqueous diluent is added to the lyophilized pharmaceutical composition to achieve the desired concentration of the components (eg, the desired concentration of active agent, trehalose, and / or buffer).

[0238] A lyophilized pharmaceutical composition comprising trehalose and active agent in a weight ratio of 1:3 to 40:1 corresponds, after reconstitution with an appropriate amount of an aqueous diluent, to a reconstituted pharmaceutical composition comprising 5 to 250 mg / mL active agent and 8.5% w / v to 20% w / v trehalose.

[0239] In some embodiments, the weight ratio of trehalose to active agent in the lyophilized pharmaceutical composition is 1:1 to 7.5:1, 1:1 to 5:1, or 2.1:1 to 4.5:1.

[0240] In some embodiments, the weight ratio of trehalose to active agent in the lyophilized pharmaceutical composition is 0.7:1 to 3.3:1 or 0.8:1 to 3:1.

[0241] In some embodiments, the weight ratio of trehalose to active agent in the lyophilized pharmaceutical composition is about 2.7:1, or about 1.4:1, or about 0.9:1.

[0242] As mentioned above, trehalose is a disaccharide composed of two glucose units linked by a 1,1-glycosidic bond. Generally, the glucose units present in trehalose are α-glucose units. Trehalose is also known as α,α-trehalose, α-D-pyranosyl-(1→1)-α-D-pyranoglucoside, α-D-pyranosyl-α-D-pyranoglucoside, and D-(+)-trehalose. The CAS number of trehalose is 6138-23-4. Trehalose has the following chemical structure:

[0243]

[0244] The forms of trehalose include anhydrous and dihydrate forms. The molar mass of trehalose in the anhydrous form is 342.3 g / mol, and the molar mass of trehalose in the dihydrate form is 378.3 g / mol.

[0245] Trehalose is available from a variety of suppliers, such as Pfanstiel (www.pfanstiehl.com). Trehalose dihydrate is also available from Sigma-Aldrich, Merck, Fisher Scientific, Acros, and Alfa Aesar.

[0246] The lyophilized pharmaceutical composition comprises a buffer. Any suitable buffer can be used. Some examples of buffers include phosphate buffers (e.g., sodium dihydrogen phosphate, disodium phosphate), amino acids such as histidine buffers (e.g., histidine hydrochloride), citrate buffers (e.g., sodium citrate), tris (2-amino-2 (hydroxymethyl) propane-1,3-diol) and acetate buffers (e.g., sodium acetate). In some embodiments, the buffer is tris or a phosphate buffer. In some embodiments, the buffer is a phosphate buffer, for example, it can be a mixture of an acidic and alkaline form of phosphate. In some embodiments, the buffer is sodium phosphate, for example, a mixture of sodium dihydrogen phosphate and disodium phosphate.

[0247] In some embodiments, the weight ratio of buffer to active agent is from 1:420 to 3:1, or from 1:200 to 1:1.8, or from 1:26 to 1:40.

[0248] In some embodiments, the buffer is sodium phosphate, and the weight ratio of sodium phosphate to active agent is 1:3 to 1:1000, or 1:3 to 1:200, or 1:5 to 1:100.

[0249] In some embodiments, the buffer is sodium phosphate, and the weight ratio of sodium phosphate to active agent is about 0.03:1.

[0250] In some embodiments, the lyophilized pharmaceutical composition comprises a surfactant. When a surfactant is included, it can be, for example, an ionic surfactant (e.g., a cationic, anionic, or zwitterionic) or a nonionic (e.g., neutral) surfactant. Some examples of surfactants include polyoxyethylene (20) sorbitan monolaurate (e.g., available under the trade name polysorbate Twain Sorbitan monooleate (e.g., sold under the trade name and Twain ), polyethylene glycols, and poloxamers (e.g., copolymers of poly(propylene oxide) and poly(ethylene oxide)) (e.g., sold under the trade name Pluronic In some embodiments, the lyophilized pharmaceutical composition comprises a surfactant which is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

[0251] The lyophilization process generally involves multiple steps, such as a freezing step, and one or more drying steps (such as a primary drying step involving sublimation and a secondary drying step involving desorption).

[0252] The lyophilized pharmaceutical composition may, for example, comprise one or more lyophilizing excipients. Some examples of lyophilizing excipients include cryoprotectants and lyoprotectants. Some examples of lyophilizing excipients include sugars (e.g., sucrose, mannitol, and dextrose), polymer excipients (e.g., polyvinyl pyrrolidone), and amino acids (e.g., glycine).

[0253] In some embodiments, the lyophilized pharmaceutical composition does not contain any additional lyophilization excipients (ie, other than the active agent, trehalose, buffer, and surfactant (if present)).

[0254] As described above, in use, an appropriate amount of aqueous diluent is added to the lyophilized pharmaceutical composition to achieve the desired component concentration (e.g., the desired concentration of the active agent, trehalose and / or buffer). The aqueous diluent may be, for example, water. Sterile high-purity water, such as water for injection, is generally used.

[0255] An amount of aqueous diluent may be added so that the concentration of the active agent in the reconstituted pharmaceutical composition is typically between 5 mg / mL and 250 mg / mL, or, for example, between 20 mg / mL and 120 mg / mL, or about 40 mg / mL, or about 80 mg / mL, or about 120 mg / mL.

[0256] As another example, an amount of aqueous diluent may be added so that the concentration of trehalose in the reconstituted pharmaceutical composition is typically at least 8.5% w / v, e.g., 8.5% w / v to 20% w / v, or 8.5% w / v to 15% w / v, or 9% w / v to 13% w / v, or 10% w / v to 12% w / v, or about 10.9% w / v.

[0257] As yet another example, an amount of aqueous diluent can be added so that the concentration of the buffer is typically up to 100 mM, e.g., up to 50 mM, or up to 20 mM, or at least 5 mM, or between 5 mM and 50 mM, or between 5 mM and 20 mM, or about 5 mM, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, or about 50 mM.

[0258] Typically, after reconstitution, the pH of the reconstituted lyophilized pharmaceutical composition is 6.5 to 8.0, for example, 6.5 to 7.0, 7.0 to 7.5, 7.5 to 8.0, 7.2 to 7.8, 7.3 to 7.7, or 7.4 to 7.6. In some embodiments, the pH of the reconstituted lyophilized pharmaceutical composition is about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, or about 8.0.

[0259] The above discussion regarding aqueous pharmaceutical compositions and the nature and amounts of required and optional characteristics (e.g., active agent, trehalose, buffer, surfactant, pH, osmolarity, and tonicity agent) also applies appropriately to lyophilized pharmaceutical compositions and reconstituted lyophilized pharmaceutical compositions.

[0260] Composition preparation

[0261] The pharmaceutical compositions of the present disclosure can be prepared by any suitable method. For example, the aqueous solution of purified soluble VEGFR-3 capture molecules can be subjected to a buffer exchange treatment step, filtered and / or mixed with other excipients as desired.

[0262] In some embodiments, an aqueous pharmaceutical composition according to the present disclosure can be prepared by mixing an aqueous solution of a soluble VEGFR-3 capture molecule with an aqueous solution of trehalose, followed by ultrafiltration-diafiltration of the resulting mixture with a buffer comprising trehalose and sodium phosphate, and then mixing with a surfactant (e.g., polyoxyethylene (20) monolaurate).

[0263] In the case of a lyophilized formulation, an aqueous pharmaceutical composition as described above may be prepared and subsequently lyophilized. The aqueous composition may, for example, be subjected to low temperature conditions such that the mixture freezes, and to low pressure conditions such that water is removed by sublimation.

[0264] Composition characteristics

[0265] Soluble VEGFR-3 capture molecules have poor stability properties and tend to form dimers or other high molecular weight aggregates when stored in aqueous pharmaceutical compositions, resulting in loss of purity, activity and shortened shelf life, or requiring low temperature storage conditions.

[0266] However, as demonstrated in the following examples, aqueous pharmaceutical compositions of the present disclosure have demonstrated unexpectedly improved stability properties, with reduced dimer formation, and maintained good levels of binding activity for VEGF-C and VEGF-D over time.

[0267] In some embodiments, the pharmaceutical composition forms less than 6%, or less than 5%, or less than 4% of the dimerized active agent after storage at 25°C for a period of 2 months.

[0268] In some embodiments, after storage at 25°C for a period of 3 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5% of the dimerized active agent.

[0269] In some embodiments, after storage at 5°C for a period of 2 months, the pharmaceutical composition forms less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0270] In some embodiments, after storage at 5°C for a period of 3 months, the pharmaceutical composition forms less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0271] In some embodiments, after storage at 5°C for a period of 6 months, the pharmaceutical composition forms less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0272] In some embodiments, after storage at 5°C for a period of 12 months, the pharmaceutical composition forms less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0273] In some embodiments, after storage at 5°C for a period of 18 months, the pharmaceutical composition forms less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0274] In some embodiments, after storage at 5°C for a period of 24 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0275] In some embodiments, after storage at 5°C for a period of 30 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4% of the dimerized active agent.

[0276] In some embodiments, the pharmaceutical composition forms less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent after storage at -20°C for a period of 2 months.

[0277] In some embodiments, after storage at -20°C for a period of 3 months, the pharmaceutical composition forms less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0278] In some embodiments, after storage at -20°C for a period of 6 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0279] In some embodiments, after storage at -20°C for a period of 12 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0280] In some embodiments, after storage at -20°C for a period of 18 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0281] In some embodiments, after storage at -20°C for a period of 24 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0282] In some embodiments, after storage at -20°C for a period of 30 months, the pharmaceutical composition forms less than 10%, or less than 8%, or less than 6%, or less than 5%, or less than 4%, or less than 3%, or less than 2% of the dimerized active agent.

[0283] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 2 months at 5°C, compared to the binding activity of the composition at 0 months.

[0284] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 3 months at 5°C, compared to the binding activity of the composition at 0 months.

[0285] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 6 months at 5°C, compared to the binding activity of the composition at 0 months.

[0286] In some embodiments, the pharmaceutical composition retains at least 80% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 12 months at 5°C, compared to the binding activity of the composition at 0 months.

[0287] In some embodiments, the pharmaceutical composition retains at least 70% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 18 months at 5°C, compared to the binding activity of the composition at 0 months.

[0288] In some embodiments, the pharmaceutical composition retains at least 60% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 24 months at 5°C compared to the binding activity of the composition at 0 months.

[0289] In some embodiments, the pharmaceutical composition retains at least 60% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 30 months at 5°C compared to the binding activity of the composition at 0 months.

[0290] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 2 months at -20°C, compared to the binding activity of the composition at 0 months.

[0291] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 3 months at -20°C, compared to the binding activity of the composition at 0 months.

[0292] In some embodiments, the pharmaceutical composition retains at least 70%, or at least 80%, or at least 90% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 6 months at -20°C, compared to the binding activity of the composition at 0 months.

[0293] In some embodiments, the pharmaceutical composition retains at least 80% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 12 months at -20°C compared to the binding activity of the composition at 0 months.

[0294] In some embodiments, the pharmaceutical composition retains at least 80% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 18 months at -20°C compared to the binding activity of the composition at 0 months.

[0295] In some embodiments, the pharmaceutical composition retains at least 80% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 24 months at -20°C compared to the binding activity of the composition at 0 months.

[0296] In some embodiments, the pharmaceutical composition retains at least 80% of the binding activity to VEGF-C and / or VEGF-D after storage for a period of 30 months at -20°C compared to the binding activity of the composition at 0 months.

[0297] As defined herein, the shelf life of a pharmaceutical composition is the period of time in months during which, when stored, the extent of dimerization of the active agent is less than 10% and the binding activity to VEGF-C and / or VEGF-D remains at least 70% of the binding activity achieved at 0 months.

[0298] In some embodiments, the pharmaceutical composition has a shelf life of at least 2 months, or at least 3 months when stored at 25°C.

[0299] In some embodiments, the pharmaceutical composition has a shelf life of at least 3 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months when stored at 5°C.

[0300] In some embodiments, the pharmaceutical composition has a shelf life of at least 3 months, or at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months when stored at -20°C.

[0301] In some embodiments, the pharmaceutical composition remains physically stable, ie, there is no apparent phase separation or precipitation of solid material, for storage at 25°C for at least 3 months.

[0302] In some embodiments, the pharmaceutical composition remains physically stable, i.e., without apparent phase separation or precipitation of solid material, for storage at 5°C for at least 3 months, at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months.

[0303] In some embodiments, the pharmaceutical composition remains physically stable, i.e., without apparent phase separation or precipitation of solid material, for storage at -20°C for at least 3 months, at least 6 months, or at least 12 months, or at least 18 months, or at least 24 months, or at least 30 months.

[0304] Therapeutic uses and methods

[0305] The pharmaceutical compositions of the present disclosure can be used to treat diseases and / or conditions for which inhibition of the interaction of VEGF-C and / or VEGF-D with the VEGFR-3 receptor provides a therapeutic response. For example, the pharmaceutical compositions of the present disclosure can be used to inhibit neovascularization and can be used to treat diseases and / or conditions associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis.

[0306] Neovascularization is the formation of new blood vessels. Angiogenesis is the formation of new blood vessels from existing ones and plays a role in a range of diseases including cancer and eye disorders such as age-related macular degeneration. Lymphangiogenesis is the formation of lymphatic vessels from pre-existing ones, and excessive lymphangiogenesis is associated with a range of diseases including edema, tumor metastasis, and lymphangiomatosis.

[0307] Therefore, the present disclosure also provides a method of inhibiting neovascularization in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein.

[0308] The present disclosure also provides a method for treating and / or preventing a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition as defined herein. Also provided is the use of a soluble VEGFR-3 capture molecule for preparing a pharmaceutical composition as defined herein, which is used to treat and / or prevent a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis. Also provided herein is a pharmaceutical composition for treating and / or preventing a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis as defined herein.

[0309] In some embodiments, the disease or condition is an ocular disease or condition. In some embodiments, the ocular disease or condition is selected from macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion, and macular telangiectasia. In some embodiments, the ocular disease or condition is wet age-related macular degeneration. In some embodiments, the ocular disease or condition is diabetic macular edema.

[0310] Pharmaceutical compositions comprising soluble VEGFR-3 capture molecules can also be used to treat other diseases and / or conditions associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis. In some embodiments, the disease or condition is cancer, such as colorectal cancer, lung cancer, breast cancer, glioblastoma, ovarian cancer, cervical cancer, and renal cancer.

[0311] Application

[0312] The pharmaceutical composition can be administered by any suitable route, eg, a route that is compatible with the disease or condition to be treated.

[0313] In some embodiments, for example, when the disease or disorder is an ocular condition, the pharmaceutical composition can be administered intravitreally. In some embodiments, the pharmaceutical composition is administered by intravitreal injection. Intravitreal injection involves administration into the vitreous humor of the eye.

[0314] In some embodiments, the pharmaceutical composition is administered using an implant device that is implanted in the eye and allows for controlled release of the active agent into the vitreous of the eye.

[0315] In some embodiments, the pharmaceutical composition is administered using a port device implanted in the eye that includes a reservoir for the pharmaceutical composition and allows for controlled release of the active agent into the vitreous of the eye.

[0316] In some embodiments, the pharmaceutical composition can be administered intravenously. In some embodiments, the pharmaceutical composition can be administered subcutaneously. In some embodiments, the pharmaceutical composition can be administered intramuscularly. In some embodiments, the pharmaceutical composition can be administered intrathecally.

[0317] In some embodiments, the pharmaceutical composition is administered by injection. In some embodiments, the pharmaceutical composition is administered by infusion.

[0318] Thus, the pharmaceutical composition can be formulated for injection (eg, for intravitreal, subcutaneous, intravenous, intramuscular, or intrathecal injection), or for administration using an ocular implant device.

[0319] In use, a suitable dose and dosing regimen of a pharmaceutical composition comprising a soluble VEGFR-3 capture molecule is used. The amount and frequency of administration may depend on a variety of factors, including the type of disease or condition, whether the active agent is administered for preventive or therapeutic purposes, the age, weight, sex and health of the person to be treated, the route of administration, and whether the soluble VEGFR-3 capture molecule is administered in combination with other active agents. In addition, pharmacogenomic information about a particular patient (the effect of genotype on pharmacokinetics, pharmacodynamics or efficacy characteristics of treatment) may affect the dose used.

[0320] Dosage form

[0321] The pharmaceutical composition can be administered in a variety of dosage forms. Therefore, the present disclosure also includes a container containing the pharmaceutical composition. The present disclosure also includes a kit, which includes a container containing the pharmaceutical composition and, optionally, instructions for administering the pharmaceutical composition to a subject.

[0322] For example, in the case of an aqueous pharmaceutical formulation, the pharmaceutical composition can be provided in a vial or bottle and administered using a syringe. In another example, the aqueous pharmaceutical composition can be provided in a pre-filled syringe. Thus, in some embodiments, the kit includes a device for administering the pharmaceutical composition.

[0323] When multiple doses are to be delivered (e.g., to the same subject or to different subjects over time), for convenience, for example, a container comprising multiple separate portions, each portion comprising a unit dose of the pharmaceutical composition (e.g., a separate bottle or a container with multiple holes) may be used. Alternatively, a kit comprising multiple containers, each containing a unit dose of the pharmaceutical composition may be used.

[0324] As described above, in some embodiments, the pharmaceutical composition may be administered using an implantable eye port device. Thus, a port device for implantation into the eye is also provided, the port device comprising a reservoir containing a pharmaceutical composition as defined herein, and wherein the port device allows controlled release of the active agent into the vitreous of the eye.

[0325] In use, the port device may be implanted, for example, through the surface of the eye (eg, through the sclera) such that it can release the active agent into the vitreous, but at least a portion of the device may be accessible for refilling the reservoir.

[0326] In some embodiments, the port device may include a reservoir connected to a membrane, an opening, a diffusion barrier, a diffusion mechanism and / or a porous structure for controlled release of an active agent. For example, it may include a semipermeable membrane, such as a titanium-containing semipermeable membrane, that allows passive diffusion of the active agent into the vitreous body of the eye.

[0327] The port device will typically include one or more retaining elements for holding the device in place (eg, through the sclera).The port device may, for example, extend through the sclera but be covered by the conjunctiva.

[0328] The port device may be refillable, for example it may comprise a refill element, such as a septum (eg, a silicone septum) that allows the reservoir to be refilled with additional drug composition using a needle.

[0329] In some embodiments, the port device is configured to receive an amount of the pharmaceutical composition sufficient to deliver a therapeutic dose of the active agent for up to 2 weeks, up to 3 weeks, up to 4 weeks, up to 1 month, up to 2 months, up to 3 months, up to 4 months, up to 5 months, or up to 6 months.

[0330] Some examples of port devices are disclosed in WO2012 / 019176, WO2012 / 065006 and WO2014 / 152959, the contents of each of which are incorporated herein by reference in their entirety.

[0331] Also provided herein is a kit comprising (i) a port device for implantation in an eye, the port device comprising a reservoir for containing a pharmaceutical composition as defined herein, wherein the port device allows controlled release of an active agent into the vitreous of the eye; and (ii) a container comprising an amount of a pharmaceutical composition as defined herein, which is used to fill the port device. In some embodiments, the kit comprises a syringe for filling the port device with the pharmaceutical composition. In some embodiments, the container comprising the pharmaceutical composition is a syringe for filling the port device.

[0332] As described above, in the case of a lyophilized pharmaceutical composition, in use, the lyophilized pharmaceutical composition can be reconstituted by mixing with an aqueous diluent prior to administration. Therefore, a kit for reconstitution is also provided, comprising a lyophilized pharmaceutical composition as defined herein and an aqueous diluent. The aqueous diluent can, for example, be sterile water, such as water for injection.

[0333] Combination therapy

[0334] While in some embodiments, the soluble VEGFR-3 capture molecules can be administered via the pharmaceutical compositions of the present disclosure as a monotherapy, in other embodiments, they are administered as part of a combination therapy regimen, e.g., in combination with additional active agents, e.g., additional active agents for the treatment and / or prevention of diseases or conditions associated with abnormal neovascularization, angiogenesis, and / or lymphangiogenesis.

[0335] The pharmaceutical composition comprising a soluble VEGFR-3 capture molecule can be administered, for example, simultaneously, sequentially or separately with another active agent. For example, a course of treatment can be prescribed for a patient, which involves administering a soluble VEGFR-3 capture molecule at certain time points and one or more additional active agents at different time points.

[0336] The additional active agent may, for example, be an additional active agent useful in the treatment and / or prevention of an ocular disease or disorder, for example an ocular disease or disorder selected from macular degeneration (e.g. wet age-related macular degeneration), diabetic retinopathy, macular edema (e.g. diabetic macular edema), retinal vein occlusion and macular telangiectasia.

[0337] In some embodiments, the additional active agent is an anti-VEGF-A agent or an anti-VEGF-B agent. Some examples of anti-VEGF-A agents and / or anti-VEGF-B agents include ranibizumab Aflibercept Bevacizumab and brolucizumab

[0338] In some embodiments, the additional active agent is pegaptanib

[0339]

[0340] In some embodiments, the additional active agent is a steroid, such as triamcinolone acetonide, dexamethasone or fluocinolone acetonide

[0341] In some embodiments, a pharmaceutical composition comprising a soluble VEGFR-3 capture molecule is administered in combination with photodynamic therapy. Photodynamic therapy involves the administration of a photosensitive active agent (e.g., verteporfin) in combination with laser therapy. In some embodiments, the pharmaceutical composition comprising a soluble VEGFR-3 capture molecule is administered in combination with laser photocoagulation therapy. Laser photocoagulation therapy involves directing a concentrated beam of high-energy laser light onto the retina to seal leaky blood vessels.

[0342] In some embodiments, a pharmaceutical composition comprising a soluble VEGFR-3 capture molecule is administered in combination with focal-grid macular laser surgery.

[0343] For example, a pharmaceutical composition of the present disclosure can be packaged together, for example, with another pharmaceutical composition comprising an additional active agent, such as in a pack or kit comprising both agents.

[0344] All publications and patents mentioned herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the event of a conflict, the present application, including any definitions herein, will control.

[0345] Sequence Listing

[0346]

[0347]

[0348]

[0349]

[0350]

[0351]

[0352] Example

[0353] The present disclosure is further illustrated by the following non-limiting examples.

[0354] Example 1: Initial Formulation Screening and DOE (Design of Experiments) Study

[0355] Formulation studies were performed to determine the preferred formulation. pH, buffers and excipients were screened and a Design of Experiments (DOE) was performed.

[0356] Briefly, initial high throughput screening was performed using one or more of Differential Scanning Calorimetry / Fluorimetry (DSC / F) and Dynamic Light Scattering (DLS). Subsequent experiments focused on Size Exclusion Chromatography (SEC) and ELISA (VEGF-D) to assess the stability of the formulation.

[0357] Results from the pH / buffer screening showed that the thermal unfolding temperature in the buffer increased from pH 6.5 to 8.0, indicating improved thermal stability.

[0358] Results from the initial excipient screening revealed that sodium and trehalose had higher thermal stability in citrate, phosphate, and tris buffers.

[0359] An accelerated stability study conducted by SEC over a 1-week period in the same buffer with a panel of excipients found that tris in phosphate buffer with mannitol, trehalose, and proline showed the least amount of degradation.

[0360] Surfactant studies showed that OPT-302 had minimal total aggregate and dimer formation when formulated in 20 mM tris buffer at pH 8.0 when trehalose and proline were excipients, and incorporation of PS-20 (polysorbate 20) into the formulation had minimal impact on stability.

[0361] The results of DOE solution stability study found that the stability of OPT-302 formulated in tris buffer increased with increasing pH and trehalose concentration.

[0362] From the in-design formulations and off-design formulations in the DOE study, the following formulations were identified as possible candidates for additional studies:

[0363] - 40 mg / mL OPT-302 in 20 mM tris, pH 8.0, 18.1% w / v trehalose, 0.01% w / v PS-20 with or without 100 mM proline;

[0364] - 40 mg / mL OPT-302 in 20 mM phosphate buffer, pH 7.5, 18.1% trehalose, and 0.01% w / v PS-20.

[0365] Example 2: Effects of trehalose content and pH on the stability of OPT-302 formulations

[0366] Formulations of OPT-302 were prepared and analyzed for their tendency to form active ingredient dimers over time. The formulations contained 40 mg / mL OPT-302, water, 10 mM sodium phosphate buffer, and 0.01% polysorbate 20. The pH of the formulations was 7.50 or 7.20 and contained 9.0% w / v, 13.6% w / v, or 18.1% w / v trehalose (prepared using 10% w / v, 15% w / v, and 20% w / v trehalose dihydrate, respectively).

[0367] The preparations were incubated at 37°C for 1 week and analyzed by SE-HPLC. The results showed that dimerization decreased with increasing trehalose content.

[0368]

[0369] Example 3: Effects of trehalose content and sodium chloride content on the stability of OPT-302 formulations

[0370] Formulations of OPT-302 were prepared and analyzed for their tendency to form high molecular weight species (e.g., dimers) of the active ingredient over time. The formulations contained 40 mg / mL OPT-302, water, and 10 mM sodium phosphate buffer (pH 7.4). The formulations contained 4.5% w / v, 6.8% w / v, 9.0% w / v, or 18.1% w / v trehalose (prepared using 5% w / v, 7.5% w / v, 10% w / v, and 20% w / v trehalose dihydrate, respectively), and 40 mM, 100 mM, or 140 mM sodium chloride.

[0371] The preparations were incubated at 37 °C for up to 2 weeks and analyzed by SE-HPLC. Figure 1 The dimerization rate was highest for the formulation containing lower concentration of trehalose.

[0372] Example 4: Stability of Additional OPT-302 Formulations

[0373] The formulation was prepared starting with OPT-302 in 40 mM NaCl, 10 mM phosphate pH 7.2 and concentrated to 54.5 mg / mL using a 50 kDa membrane.

[0374] OPT-302 was purified using a Superdex S200 column loaded at approximately 3.2% of column volume (CV) and run at 0.5 CV / hour in 40 mM NaCl, 10 mM phosphate pH 7.2. S200 fractions with ≥97% monomer were pooled and 7.5% trehalose was added to the S200 purification pool containing 37.5% trehalose stock solution prior to pre-formulation for TFF. The % monomer determined by SE-UPLC was 98.7%.

[0375] One fifth of the stock solution was then added to 6.8% trehalose and concentrated to 59 g / L before adding polysorbate 20 (0.03%) and diluting to 40 g / L with 6.85% w / v trehalose, 0.03% v / v PS20, 10 mM Phos, 40 mM NaCl, pH 7.2.

[0376] Three-fifths of the stock solution was diafiltered with 9.0% w / v trehalose, 10 mM Phos, pH 7.5 (>7 DV) and then divided into three parts. The first part was diluted to 40 mg / mL with 9.0% w / v trehalose, 10 mM phosphate pH 7.5. The second part was added to 13.6% trehalose using 40% trehalose and then diluted to 40 mg / mL OPT-302 to reach 13.6% w / v trehalose, 10 mM Phos, pH 7.5, and then PS-20 (0.01%) was added. The third part was added to 18.1% trehalose using 40% trehalose and then diluted to 40 mg / mL OPT-302 to reach 18.1% w / v trehalose, 10 mM Phos, pH 7.5, and then PS-20 (0.01%) was added.

[0377] The final fifth of the stock was diafiltered against 13.6% w / v trehalose, 20 mM Tris, pH 8.0, (>7 DV), diluted to 40 mg / mL OPT-302 in the same buffer, and then PS20 (0.01%) was added.

[0378] All preparations were then subjected to SE-UPLC and the results are shown in the table below.

[0379]

[0380] As can be seen from the table, the formulations concentrated in the presence of sodium chloride had the lowest monomer stability. Those formulations that were diafiltered to remove the sodium chloride had improved stability, with the % monomer being higher for those formulations containing higher amounts of trehalose.

[0381] Example 5: Long-term and accelerated stability studies

[0382] OPT-302 formulations were prepared as follows, all containing 40 mg / mL OPT302:

[0383] 1. A lyophilized formulation comprising 6.8% w / v trehalose (prepared using 7.5% w / v trehalose dihydrate), 0.03% w / v PS20, 10 mM sodium phosphate, 40 mM NaCl, pH 7.2.

[0384] 2. An aqueous formulation comprising 6.8% w / v trehalose (prepared using 7.5% w / v trehalose dihydrate), 0.03% PS 20, 10 mM sodium phosphate, 40 mM NaCl, pH 7.2.

[0385] 3. An aqueous formulation comprising 9.0% w / v trehalose (prepared using 10% w / v trehalose dihydrate), 0.01% PS 20, 10 mM sodium phosphate, pH 7.5.

[0386] 4. An aqueous formulation comprising 13.6% w / v trehalose (prepared using 15% w / v trehalose dihydrate), 0.01% PS 20, 10 mM sodium phosphate, pH 7.5.

[0387] 5. An aqueous formulation comprising 18.1% w / v trehalose (prepared using 20% ​​w / v trehalose dihydrate), 0.01% PS 20, 10 mM sodium phosphate, pH 7.5.

[0388] 6. An aqueous formulation comprising 13.6% w / v trehalose (prepared using 15% w / v trehalose dihydrate), 0.01% PS 20, 20 mM Tris, pH 8.

[0389] The formulations were set at 25°C (monthly testing for 3 months, SEC) and 5°C (quarterly testing for 24 months by SEC). The summary results of SEC are in Figure 2 and Figure 3 Available in.

[0390] The accelerated results at 25°C showed that trehalose concentrations above 6.8% w / v substantially stabilized the monomer content and indicated that formulations containing high trehalose concentrations without added sodium chloride may be stable at 5°C.

[0391] For real-time conditions (5°C, storage for 24 months), the monomer content of formulations containing more than 6.8% w / v trehalose all had significantly higher stability than the comparative formulations at 24 months.

[0392] In summary, in liquid form, long-term refrigerated storage (15 months) and accelerated stability results indicate that OPT-302 can be reconstituted at higher trehalose concentrations, which results in superior stability.

[0393] Example 6: OPT-302 Formulation

[0394] A preferred aqueous formulation of OPT-302 was identified, comprising the following components:

[0395]

[0396] Preparation density = 1.041 kg / L

[0397] An additional comparative OPT-302 formulation had the following components:

[0398]

[0399] The comparative formulations were found to form high levels of OPT-302 dimers upon storage, as demonstrated by Example 8 below.

[0400] Example 7: Preparation of OPT-302 formulation

[0401] An aliquot of an aqueous solution (trehalose spike) containing a high concentration of trehalose (37.5% w / v trehalose dihydrate, 10 mM sodium phosphate, pH 7.5) was added to a virus filtration pool (virus filtration pool) containing OPT-302 to achieve a concentration of 12% w / v trehalose dihydrate and mixed for at least 10 minutes to provide a mixture (conditioned virus filtration pool).

[0402] The mixture was filtered through a 0.2 μm filter and ultrafiltered, diafiltered, and concentrated using tangential flow filtration using equilibration, diafiltration, and wash buffers containing 12% w / v trehalose dihydrate, 10 mM sodium phosphate, pH 7.5 to provide a mixture with a target OPT-302 concentration of 46 to 55 mg / mL (UFDF pool).

[0403] The concentration of OPT-302 in the UFDF pool was determined, and an amount of dilution buffer (12% w / v trehalose dihydrate, 10 mM sodium phosphate, pH 7.5) was added to the UFDF pool to obtain an OPT-302 concentration of approximately 44 g / L (diluted UFDF pool). The diluted UFDF pool was mixed for at least 30 minutes using a Wave Mixer.

[0404] The target weight of formulation buffer (12% w / v trehalose dihydrate, 10 mM sodium phosphate, polyoxyethylene (20) sorbitan monolaurate, pH 7.5) that needs to be added to the diluted UFDF pool to obtain a concentration of 0.01% (w / v) polyoxyethylene (20) sorbitan monolaurate was calculated and added. The formulated diluted UFDF pool was mixed for at least 15 minutes using a Wave Mixer.

[0405] The expected concentration of OPT-302 is 36 to 44 mg / mL.

[0406] Example 8: Stability of OPT-302 Formulation

[0407] Three OPT-302 formulations were prepared with the following components:

[0408] - Formulation 1: 41.5 mg / mL OPT-302, 10 mM sodium phosphate, 12% w / v trehalose dihydrate (10.9% w / v trehalose), 0.01% w / v polyoxyethylene (20) sorbitan monolaurate, WFI, pH 7.5.

[0409] - Comparative Formulation 1: 41 mg / mL OPT-302, 10 mM sodium phosphate, 40 mM sodium chloride, 7.5% w / v trehalose dihydrate (6.8% w / v trehalose), 0.03% w / v polyoxyethylene (2) sorbitan monolaurate, WFI, pH 7.2;

[0410] - Comparative Formulation 2: 42.4 mg / mL OPT-302, 10 mM sodium phosphate, 40 mM sodium chloride, 7.5% w / v trehalose dihydrate (6.8% w / v trehalose), 0.03% w / v polyoxyethylene (2) sorbitan monolaurate, pH 7.2, WFI;

[0411] The formulations were stored at various temperatures and the stability characteristics of the formulations were determined. Data for Formulation 1 stored at -20°C, 5°C, 25°C and 40°C and data for the comparative formulation stored at 25°C and 40°C are shown below.

[0412] Preparation 1 at -20°C

[0413]

[0414] Preparation 1 at 5°C

[0415]

[0416]

[0417] Preparation 1 at 25°C

[0418]

[0419] Preparation 1 at 40°C

[0420]

[0421] Comparative Preparation 1 at 25°C

[0422]

[0423] Comparative Preparation 1 at 40°C

[0424]

[0425] Comparative Formulation 2 at 25°C

[0426]

[0427]

[0428] Comparative Preparation 2 at 40°C

[0429]

[0430] Compared to Comparative Formulations 1 and 2, Formulation 1 had lower levels of high molecular weight species formation over time, especially at higher temperature conditions.

[0431] Formulation 1 was also shown to have good activity in binding VEGF-C and VEGF-D as determined by ELISA.

[0432] ELISA method

[0433] The assay plate was coated overnight with 0.1 μg / mL of VEGF-C ligand or 1.0 μg / mL of VEGF-D ligand. The OPT-302 reference standard and test sample were diluted to a starting concentration of 1500 ng / mL and a series of 2-fold dilutions were performed, including 1.5 ng / mL to 1500 ng / mL. The plate was incubated at 25°C for 60 minutes and then washed to remove unbound samples. The bound molecules were detected by adding HRP-conjugated rabbit anti-human IgG to the assay plate and incubated at 25°C for 60 minutes. TMB substrate was then added and the assay plate was incubated in the dark at room temperature for 10 minutes. The color development was stopped by adding 1M HCl stop solution and detected by absorbance at 450nm. The intensity of the yellow color is proportional to the amount of the bound OPT-302 molecule, which in turn reflects the activity of the OPT-302 reference standard or test sample. A 4-parameter curve was then generated by plotting the mean value of log10 for the series of 2-fold dilution concentrations. The reportable value is the relative potency (%) of the test sample, ie, the ratio of the EC50 of the reference standard to the EC50 of the test sample.

[0434] When stored at -20°C, Formulation 1 was found to have the following activities: 0 month, VEGF-C: 107%, VEGF-D: 102%; 0.5 month, VEGF-C 126%, VEGF-D 116%; 1 month, VEGF-C: 118%, VEGF-D: 100%; 2 months, VEGF-C: 108%, VEGF-D: 106%.

[0435] When stored at 5°C, Formulation 1 was found to have the following activities: 0 month, VEGF-C: 107%, VEGF-D: 102%; 0.5 month, VEGF-C 114%, VEGF-D 112%; 1 month, VEGF-C: 115%, VEGF-D: 101%; 2 months, VEGF-C: 107%, VEGF-D: 105%.

[0436] Example 9: Stability of OPT-302 Formulation

[0437] A batch of a preferred aqueous formulation for OPT-302 was prepared containing the following components:

[0438]

[0439] Part of the formulation batches were stored at -20±5°C and 5±3°C for long periods of time and the stability characteristics of the formulations were determined.

[0440] When stored at -20°C or 5°C for up to 24 months, key stability assays (e.g., SE-UPLC) showed minimal changes in monomer content, decreasing by 0.8% from the initial amount at -20°C and 3% at 5°C, while high molecular weight (dimer) increased by 0.7% at -20°C and 2.2% at 5°C compared to the start of the stability study. Similarly, the binding activity of VEGF-C and VEGF-D measured by ELISA was minimally reduced by 9% to 19%.

[0441] The formulation has excellent storage stability properties at -20°C and 5°C for a specific period of time.

[0442] Another batch of OPT-302 formulated as described above was similarly stored at 5±3°C for 24 months and subsequently characterized by mass spectrometry analysis for isoform variants. It was found that during this period, the deamidated variant increased by 9%, the isomerized variant decreased by 9%, and the change in the oxidized variant was negligible. Therefore, during the long-term (24-month) storage period, the change in the charged variant was overall minimal, indicating the retention of the molecular structure in the formulation.

Claims

1. An aqueous pharmaceutical composition comprising: an active agent which is a soluble VEGFR-3 capture molecule, said active agent being present at a concentration of 5 mg / mL to 250 mg / mL; Trehalose; Buffering agents; and water; wherein the pH of the aqueous pharmaceutical composition is 6.5 to 8.0; And wherein the pharmaceutical composition comprises trehalose at a concentration of at least 7.0% w / v and / or the pharmaceutical composition does not comprise added sodium chloride.

2. The aqueous pharmaceutical composition of claim 1, wherein the pharmaceutical composition does not comprise added sodium chloride.

3. An aqueous pharmaceutical composition according to claim 1 or 2, wherein the pharmaceutical composition comprises trehalose at a concentration of at least 7.0% w / v.

4. An aqueous pharmaceutical composition according to any one of claims 1 to 3, wherein trehalose is present in a concentration of up to 20% w / v.

5. The aqueous pharmaceutical composition of claim 3, wherein trehalose is present in a concentration of 8.5% w / v to 15% w / v.

6. The aqueous pharmaceutical composition of claim 5, wherein the composition comprises about 10.9% w / v trehalose.

7. An aqueous pharmaceutical composition according to any one of claims 1 to 6, wherein the soluble VEGFR-3 capture molecule comprises a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain.

8. The aqueous pharmaceutical composition of claim 7, wherein the ligand-binding polypeptide comprises an amino acid sequence defined by positions 25 to 329 of SEQ ID NO: 1, provided that the positions of the polypeptide corresponding to positions 104 to 106 of SEQ ID NO: 1 are not identical to NXS or NXT; wherein the ligand-binding polypeptide retains four N-glycosylation sequence stretch sites corresponding to positions 33 to 35 of SEQ ID NO: 1, positions 166 to 168 of SEQ ID NO: 1, positions 251 to 253 of SEQ ID NO: 1, and positions 299 to 301 of SEQ ID NO: 1, and is glycosylated at the four N-glycosylation sequence stretch sites.

9. The aqueous pharmaceutical composition of claim 7 or claim 8, wherein the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99 to 330 of SEQ ID NO:

2.

10. An aqueous pharmaceutical composition according to claim 8 or 9, wherein the soluble VEGFR-3 capture molecule has an amino acid sequence shown in any one of SEQ ID NOs: 3 to 6, or has an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 3, or has an amino acid sequence defined by positions 1 to 536 of SEQ ID NO: 4, or has an amino acid sequence defined by positions 1 to 546 of SEQ ID NO: 5, or has an amino acid sequence defined by positions 1 to 546 of SEQ ID NO:

6.

11. An aqueous pharmaceutical composition according to claim 7, wherein the ligand-binding polypeptide comprises an amino acid sequence defined by positions 25 to 329 of SEQ ID NO: 1; wherein the ligand-binding polypeptide retains five N-glycosylation sequence segment sites corresponding to positions 33 to 35 of SEQ ID NO: 1, positions 104 to 106 of SEQ ID NO: 1, positions 166 to 168 of SEQ ID NO: 1, positions 251 to 253 of SEQ ID NO: 1, and positions 299 to 301 of SEQ ID NO: 1, and is glycosylated at the five N-glycosylation sequence segment sites.

12. The aqueous pharmaceutical composition of claim 7 or claim 11, wherein the immunoglobulin constant domain fragment comprises the amino acid sequence defined by positions 99 to 330 of SEQ ID NO:

2.

13. The aqueous pharmaceutical composition of claim 12, wherein the soluble VEGFR-3 capture molecule has the amino acid sequence shown in SEQ ID NO:7, or has the amino acid sequence defined by positions 1 to 547 of SEQ ID NO:

7.

14. The aqueous pharmaceutical composition of any one of claims 1 to 13, wherein the active agent is present in a concentration of up to 120 mg / mL.

15. The aqueous pharmaceutical composition of any one of claims 1 to 14, wherein the active agent is present at a concentration of about 40 mg / mL, or about 80 mg / mL, or about 120 mg / mL.

16. The aqueous pharmaceutical composition of any one of claims 1 to 15, wherein the pH of the composition is 7.2 to 7.

8.

17. The aqueous pharmaceutical composition of claim 16, wherein the pH of the composition is about 7.

5.

18. The aqueous pharmaceutical composition of any one of claims 1 to 17, wherein the buffer is sodium phosphate.

19. The aqueous pharmaceutical composition of any one of claims 1 to 18, wherein the buffer is present at a concentration of 5 mM to 100 mM.

20. The aqueous pharmaceutical composition of any one of claims 1 to 19, wherein the buffer is present in a concentration of up to 50 mM.

21. The aqueous pharmaceutical composition of claim 20, wherein the buffer is present at a concentration of about 10 mM.

22. An aqueous pharmaceutical composition according to any one of claims 1 to 21, wherein the composition comprises a surfactant.

23. The aqueous pharmaceutical composition of claim 22, wherein the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

24. The aqueous pharmaceutical composition of claim 23, wherein the surfactant is present at a concentration of 0.005% to 0.2% w / v.

25. The aqueous pharmaceutical composition of claim 24, wherein the surfactant is present at a concentration of about 0.01% w / v.

26. The aqueous pharmaceutical composition of any one of claims 1 to 25, wherein the osmolarity of the composition is 300 to 1000 mOsm / kg.

27. The aqueous pharmaceutical composition of claim 26, wherein the osmolarity of the composition is 350 mOsm / kg.

28. The aqueous pharmaceutical composition of claim 27, wherein the osmolarity of the composition is 400 mOsm / kg.

29. The aqueous pharmaceutical composition of claim 28, wherein the composition has an osmolarity of 400 to 600 mOsm / kg.

30. The aqueous pharmaceutical composition of any one of claims 1 to 29, wherein the composition is substantially free of sodium chloride.

31. An aqueous pharmaceutical composition according to any one of claims 1 to 30, wherein the composition comprises no additional sugars.

32. The aqueous pharmaceutical composition of any one of claims 1 to 31, wherein the composition does not comprise an additional tonicity adjusting agent.

33. An aqueous pharmaceutical composition according to any one of claims 1 to 32, wherein the composition consists essentially of: an active agent at a concentration of about 40 mg / ml, which is a soluble VEGFR-3 capture molecule comprising a ligand binding polypeptide fused to an immunoglobulin constant domain fragment, the ligand binding polypeptide comprising immunoglobulin-like domains 1 to 3 of the extracellular domain of human VEGFR-3, and optionally having one or more modifications in the N-glycan region of the extracellular domain; Trehalose at a concentration of about 10.9% w / v; Sodium phosphate at a concentration of about 10 mM; Polyoxyethylene (20) sorbitan monolaurate at a concentration of about 0.01% w / v; and water; The pH of the aqueous pharmaceutical composition is about 7.

5.

34. A lyophilized pharmaceutical composition for reconstitution, comprising: an active agent, which is a soluble VEGFR-3 capture molecule; Trehalose; and Buffers; The weight ratio of trehalose to the active agent is 1:3 to 40:

1.

35. The lyophilized pharmaceutical composition of claim 34, wherein the weight ratio of trehalose to active agent is 1:1 to 7.5:1, 1:1 to 5:1, or 2.1:1 to 4.5:

1.

36. The lyophilized pharmaceutical composition of claim 35, wherein the weight ratio of trehalose to active agent is about 2.7:

1.

37. The lyophilized pharmaceutical composition according to any one of claims 34 to 36, wherein the buffer is sodium phosphate.

38. The lyophilized pharmaceutical composition of claim 37, wherein the buffer is sodium phosphate, and the weight ratio of sodium phosphate to active agent is 1:3 to 1:1000, or 1:3 to 1:200, or 1:5 to 1:

100.

39. The lyophilized pharmaceutical composition of claim 38, wherein the buffer is sodium phosphate and the weight ratio of sodium phosphate to active agent is about 0.03:

1.

40. The lyophilized pharmaceutical composition according to any one of claims 34 to 39, wherein the composition comprises a surfactant.

41. The lyophilized pharmaceutical composition according to claim 40, wherein the surfactant is polyoxyethylene (20) sorbitan monolaurate or polyoxyethylene (20) sorbitan monooleate.

42. A reconstituted pharmaceutical composition, wherein the pharmaceutical composition is obtained by mixing the lyophilized pharmaceutical composition according to any one of claims 34 to 41 with an aqueous diluent.

43. The pharmaceutical composition of any one of claims 1 to 33 or 42, wherein the pharmaceutical composition is formulated for intravitreal injection.

44. A method of inhibiting neovascularization in a subject, comprising administering to the subject an effective amount of the pharmaceutical composition of any one of claims 1 to 33, 42 or 43.

45. A method for treating and / or preventing a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis in a subject, comprising administering to the subject an effective amount of a pharmaceutical composition according to any one of claims 1 to 33, 42 or 43.

46. ​​Use of a VEGF-C and / or VEGF-D capture molecule or a salt thereof for the preparation of a pharmaceutical composition as defined in any one of claims 1 to 33, 42 or 43 for the treatment and / or prevention of a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis.

47. A pharmaceutical composition according to any one of claims 1 to 33, 42 or 43 for use in the treatment and / or prevention of a disease or condition associated with abnormal neovascularization, angiogenesis and / or lymphangiogenesis.

48. The method of claim 45, the use of claim 38, or the pharmaceutical composition for use of claim 47, wherein the disease or condition is an ocular disease or condition.

49. The method, use or pharmaceutical composition for use according to claim 48, wherein the ocular disease or condition is selected from macular degeneration, diabetic retinopathy, macular edema, retinal vein occlusion and macular telangiectasia.

50. The method, use or pharmaceutical composition for use according to claim 49, wherein the ocular disease or disorder is wet age-related macular degeneration.

51. The pharmaceutical composition for the method, use or use of claim 49, wherein the ocular disease or disorder is diabetic macular edema.

52. The pharmaceutical composition for the method, use or use according to any one of claims 44 to 51, wherein the pharmaceutical composition is administered in combination with an additional active agent.

53. The pharmaceutical composition for the method, use or use of any one of claims 44 to 52, wherein the additional active agent is an anti-VEGF-A agent or an anti-VEGF-B agent.

54. The pharmaceutical composition for the method, use or use of claim 53, wherein the additional active agent is selected from ranibizumab, aflibercept, bevacizumab and brolucizumab.

55. The pharmaceutical composition for the method, use or use according to any one of claims 44 to 54, wherein the pharmaceutical composition is administered intravitreally.

56. A pharmaceutical composition for the method, use or application of any one of claims 44 to 55, wherein the pharmaceutical composition is administered using a port device implanted in the eye, the port device comprising a reservoir for the pharmaceutical composition and allowing controlled release of the active agent into the vitreous of the eye.

57. A port device for implantation in an eye, the port device comprising a reservoir containing a pharmaceutical composition according to any one of claims 1 to 33, 42 or 43, and wherein the port device allows controlled release of the active agent into the vitreous of the eye.

58. The port device of claim 57, wherein the port device comprises a semipermeable membrane that allows passive diffusion of the active agent into the vitreous of the eye.

59. The port device of claim 57 or 58, wherein the port device comprises a septum that allows the reservoir to be refilled with additional drug composition using a needle.

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